Method for identification, isolation and production of antigens to a specific pathogen

ABSTRACT

Described is a method for identification, isolation and production of hyperimmune serum-reactive antigens from a specific pathogen, a tumor, an allergen or a tissue or host prone to autoimmunity, said antigens being suited for use in a vaccine for a given type of animal or for humans, which is characterized by the following steps: —providing an antibody preparation from a plasma pool of said given type of animal or from a human plasma pool or individual sera with antibodies against said specific pathogen, tumor, allergen or tissue or host prone to auto-immunity, —providing at least one expression library of said specific pathogen, tumor, allergen or tissue or host prone to auto-immunity, —screening said at least one expression library with said antibody preparation, —identifying antigens which bind in said screening to antibodies in said antibody preparation, —screening the identified antigens with individual antibody preparations from individual sera from individuals with antibodies against said specific pathogen, tumor, allergen or tissue or host prone to auto-immunity, —identifying the hyperimmune serum-reactive antigen portion of said identified antigens and which hyperimmune serum-reactive antigens bind to a relevant portion of said individual antibody preparations from said individual sera and —optionally isolating said hyperimmune serum-reactive antigens and producing said hyperimmune serum-reactive antigens by chemical or recombinant methods.

This application is a U.S. national phase application under 35 U.S.C. §371 of PCT Application No. PCT/EP02/00546 filed 21 Jan. 2002, which claims priority to Austrian Application No. A 130/2001 filed 26 Jan. 2001.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING SUBMITTED ON A COMPACT DISC

The Sequence Listing of this application is submitted on one compact disc (Copy 1), together with a duplicate thereof (Copy 2), each created on Sep. 11, 2006, and each containing one 1,609 kb file entitled “SONN035.TXT.” The material contained on the compact disc is specifically incorporated herein by reference.

The invention relates to a method for identification, isolation and production of antigens to a specific pathogen as well as new antigens suitable for use in a vaccine for a given type of animal or for humans.

Vaccines can save more lives (and resources) than any other medical intervention. Owing to world-wide vaccination programmes the incidence of many fatal diseases has been decreased drastically. Although this notion is valid for a whole panel of diseases, e.g. diphtheria, pertussis, measles and tetanus, there are no effective vaccines for numerous infectious disease including most viral infections, such as HIV, HCV, CMV and many others. There are also no effective vaccines for other diseases, infectious or non-infectious, claiming the lives of millions of patients per year including malaria or cancer. In addition, the rapid emergence of antibiotic-resistant bacteria and microorganisms calls for alternative treatments with vaccines being a logical choice. Finally, the great need for vaccines is also illustrated by the fact that infectious diseases, rather than cardiovascular disorders or cancer or injuries remain the largest cause of death and disability in the world.

Several established vaccines consist of live attenuated organisms where the risk of reversion to the virulent wild-type strain exists. In particular in immunocompromised hosts this can be a live threatening scenario. Alternatively, vaccines are administered as a combination of pathogen-derived antigens together with compounds that induce or enhance immune responses against these antigens (these compounds are commonly termed adjuvant), since these subunit vaccines on their own are generally not effective.

Whilst there is no doubt that the above vaccines are valuable medical treatments, there is the disadvantage that, due to their complexity, severe side effects can be evoked, e.g. to antigens that are contained in the vaccine that display cross-reactivity with molecules expressed by cells of vaccinated individuals. In addition, existing requirements from regulatory authorities, e.g. the World Health Organization (WHO), the Food and Drug Administration (FDA), and their European counterparts, for exact specification of vaccine composition and mechanisms of induction of immunity, are difficult to meet.

Some widely used vaccines are whole cell-vaccines (attenuated bacteria or viruses (e.g. Bacille Calmette-Guerin (BCG) (tuberculosis), Measles, Mumps, Rubella, Oral Polio Vaccine (Sabin), killed bacteria or viruses (e.g. Pertussis, Inactivated polio vaccine (Salk)), subunit-vaccines (e.g. Toxoid (Diphtheria, Tetanus)), Capsular polysaccharide (H. influenzae type B), Yeast recombinant subunit (Hepatitis B surface protein).

A vaccine can contain a whole variety of different antigens. Examples of antigens are whole-killed organisms such as inactivated viruses or bacteria, fungi, protozoa or even cancer cells. Antigens may also consist of subfractions of these organisms/tissues, of proteins, or, in their most simple form, of peptides. Antigens can also be recognized by the immune system in form of glycosylated proteins or peptides and may also be or contain polysaccharides or lipids. Short peptides can be used since for example cytotoxic T-cells (CTL) recognize antigens in form of short usually 8-11 amino acids long peptides in conjunction with major histocompatibility complex (MHC). B-cells can recognize linear epitopes as short as 4-5 amino acids, as well as three dimensional structures (conformational epitopes). In order to obtain sustained, antigen-specific immune responses, adjuvants need to trigger immune cascades that involve all cells of the immune system necessary. Primarily, adjuvants are acting, but are not restricted in their mode of action, on so-called antigen presenting cells (APCs). These cells usually first encounter the antigen(s) followed by presentation of processed or unmodified antigen to immune effector cells. Intermediate cell types may also be involved. Only effector cells with the appropriate specificity are activated in a productive immune response. The adjuvant may also locally retain antigens and co-injected other factors. In addition the adjuvant may act as a chemoattractant for other immune cells or may act locally and/or systemically as a stimulating agent for the immune system.

Antigen presenting cells belong to the innate immune system, which has evolved as a first line host defence that limits infection early after exposure to microorganisms. Cells of the innate immune system recognize patterns or relatively non-specific structures expressed on their targets rather than more sophisticated, specific structures which are recognized by the adaptive immune system. Examples of cells of the innate immune system are macrophages and dendritic cells but also granulocytes (e.g. neutrophiles), natural killer cells and others. By contrast, cells of the adaptive immune system recognize specific, antigenic structures, including peptides, in the case of T-cells and peptides as well as three-dimensional structures in the case of B-cells. The adaptive immune system is much more specific and sophisticated than the innate immune system and improves upon repeated exposure to a given pathogen/antigen. Phylogenetically, the innate immune system is much older and can be found already in very primitive organisms. Nevertheless, the innate immune system is critical during the initial phase of antigenic exposure since, in addition to containing pathogens, cells of the innate immune system, i.e. APCs, prime cells of the adaptive immune system and thus trigger specific immune responses leading to clearance of the intruders. In sum, cells of the innate immune system and in particular APCs play a critical role during the induction phase of immune responses by a) containing infections by means of a primitive pattern recognition system and b) priming cells of the adaptive immune system leading to specific immune responses and memory resulting in clearance of intruding pathogens or of other targets. These mechanisms may also be important to clear or contain tumor cells.

The antigens used for such vaccines have often been selected by chance or by easiness of availability. There is a demand to identify efficient antigens for a given pathogen or—preferably—an almost complete set of all antigens of a given pathogen which are practically (clinically) relevant. Such antigens may be preferred antigen candidates in a vaccine.

It is therefore an object of the present invention to comply with these demands and to provide a method with which such antigens may be provided and with which a practically complete set of antigens of e.g. a given pathogen may be identified with a given serum as antibody source. Such a method should also be suitable for rapidly changing pathogens which evolve a fast resistance against common drugs or vaccines. The method should also be applicable to identify and isolate tumor antigens, allergens, autoimmune antigens.

Therefore, the present invention provides a method for identification, isolation and production of hyperimmune serum-reactive antigens from a specific pathogen, a tumor, an allergen or a tissue or host prone to auto-immunity, especially from a specific pathogen, said antigens being suited for use in a vaccine for a given type of animal or for humans, said method being characterized by the following steps:

-   -   providing an antibody preparation from a plasma pool of said         given type of animal or from a human plasma pool or individual         sera with antibodies against said specific pathogen, a tumor, an         allergen or a tissue or host prone to auto-immunity,     -   providing at least one expression library of said specific         pathogen, a tumor, an allergen or a tissue or host prone to         auto-immunity,     -   screening said at least one expression library with said         antibody preparation,     -   identifying antigens which bind in said screening to antibodies         in said antibody preparation,     -   screening the identified antigens with individual antibody         preparations from individual sera from individuals with         antibodies against said specific pathogen, tumor, allergen or         tissue or host prone to auto-immunity,     -   identifying the hyperimmune serum-reactive antigen portion of         said identified antigens which hyperimmune serum-reactive         antigens bind to a relevant portion of said individual antibody         preparations from said individual sera and     -   optionally isolating said hyperimmune serum-reactive antigens         and producing said hyperimmune serum-reactive antigens by         chemical or recombinant methods.

This method is also suitable in general for identifying a practically complete set of hyperimmune serum-reactive antigens of a specific pathogen with given sera as antibody sources, if at least three different expression libraries are screened in a pathogen/antigen identification programme using the method according to the present invention. The present invention therefore also relates to a method for identification, isolation and production of a practically complete set of hyperimmune serum-reactive antigens of a specific pathogen, said antigens being suited for use in a vaccine for a given type of animal or for humans, which is characterized by the following steps:

-   -   providing an antibody preparation from a plasma pool of said         given type of animal or from a human plasma pool or individual         sera with antibodies against said specific pathogen,     -   providing at least three different expression libraries of said         specific pathogen,     -   screening said at least three different expression libraries         with said antibody preparation,     -   identifying antigens which bind in at least one of said at least         three screenings to antibodies in said antibody preparation,     -   screening the identified antigens with individual antibody         preparations from individual sera from individuals with         antibodies against said specific pathogen,     -   identifying the hyperimmune serum-reactive antigen portion of         said identified antigens which hyperimmune serum-reactive         antigens bind to a relevant portion of said individual antibody         preparations from said individual sera,     -   repeating said screening and identification steps at least once,     -   comparing the identified hyperimmune serum-reactive antigens         identified in the repeated screening and identification steps         with the identified hyperimmune serum-reactive antigens         identified in the initial screening and identification steps,     -   further repeating said screening and identification steps, if at         least 5% of the hyperimmune serum-reactive antigens have been         identified in the repeated screening and identification steps         only, until less than 5% of the hyperimmune serum-reactive         antigens are identified in a further repeating step only to         obtain a complete set of hyperimmune serum-reactive antigens of         a specific pathogen and     -   optionally isolating said hyperimmune serum-reactive antigens         and producing said hyperimmune serum-reactive antigens by         chemical or recombinant methods.

The method according to the present invention mainly consists of three essential parts, namely 1. identifying hyperimmune serum sources containing specific antibodies against a given pathogen, 2. screening of suitable expression libraries with a suitable antibody preparation wherein candidate antigens (or antigenic fragments of such antigens) are selected, and—3. in a second screening round, wherein the hyperimmune serum-reactive antigens are identified by their ability to bind to a relevant portion of individual antibody preparations from individual sera in order to show that these antigens are practically relevant and not only hyperimmune serum-reactive, but also widely immunogenic (i.e. that a lot of individual sera react with a given antigen). With the present method it is possible to provide a set of antigens of a given pathogen which is practically complete with respect to the chosen pathogen and the chosen serum. Therefore, a bias with respect to “wrong” antigen candidates or an incomplete set of antigens of a given pathogen is excluded by the present method.

Completeness of the antigen set of a given pathogen within the meaning of the present invention is, of course, dependent on the completeness of the expression libraries used in the present method and on the quality and size of serum collections (number of individual plasmas/sera) tested, both with respect to representability of the library and usefulness of the expression system. Therefore, preferred embodiments of the present method are characterized in that at least one of said expression libraries is selected from a ribosomal display library, a bacterial surface library and a proteome.

A serum collection used in the present invention should be tested against a panel of known antigenic compounds of a given pathogen, such as polysaccharide, lipid and proteinaceous components of the cell wall, cell membranes and cytoplasma, as well as secreted products. Preferably, three distinct serum collections are used: 1. With very stable antibody repertoire: normal adults, clinically healthy people, who overcome previous encounters or currently carriers of e.g. a given pathogen without acute disease and symptoms, 2. With antibodies induced actually by the presence of the pathogenic organism: patients with acute disease with different manifestations (e.g. S. aureus sepsis or wound infection, etc.), 3. With no specific antibodies at all (as negative controls): 5-8 months old babies who lost the maternally transmitted immunoglobulins 5-6 months after birth. Sera have to react with multiple pathogen-specific antigens in order to consider hyperimmune for a given pathogen (bacteria, fungus, worm or otherwise), and for that relevant in the screening method according to the present invention.

In the antigen identification programme for identifying a complete set of antigens according to the present invention, it is preferred that said at least three different expression libraries are at least a ribosomal display library, a bacterial surface library and a proteome. It has been observed that although all expression libraries may be complete, using only one or two expression libraries in an antigen identification programme will not lead to a complete set of antigens due to preferential expression properties of each of the different expression libraries. While it is therefore possible to obtain hyperimmune serum-reactive antigens by using only one or two different expression libraries, this might in many cases not finally result in the identification of a complete set of hyperimmune serum-reactive antigens. Of course, the term “complete” according to the present invention does not indicate a theoretical maximum but is indeed a practical completeness, i.e. that at least 95% of the practically relevant antigens or antigenic determinants have been identified of a given pathogen. The practical relevance is thereby defined by the occurrence of antibodies against given antigens in the patient population.

According to the present invention also serum pools or plasma fractions or other pooled antibody containing body fluids are “plasma pools”.

An expression library as used in the present invention should at least allow expression of all potential antigens, e.g. all surface proteins of a given pathogen. With the expression libraries according to the present invention, at least one set of potential antigens of a given pathogen is provided, this set being preferably the complete theoretical complement of (poly-)peptides encoded by the pathogen's genome (i.e. genomic libraries as described in Example 2) and expressed either in a recombinant host (see Example 3) or in vitro (see Example 4). This set of potential antigens can also be a protein preparation, in the case of extracellular pathogens preferably a protein preparation containing surface proteins of said pathogen obtained from said pathogen grown under defined physiological conditions (see Example 5). While the genomic approach has the potential to contain the complete set of antigens, the latter one has the advantage to contain the proteins in their naturally state i.e. including for instance post-translational modifications or processed forms of these proteins, not obvious from the DNA sequence. These or any other sets of potential antigens from a pathogen, a tumor, an allergen or a tissue or host prone to auto-immunity are hereafter referred to as “expression library”. Expression libraries of very different kinds may be applied in the course of the present invention. Suitable examples are given in e.g. Ausubel et al., 1994. Especially preferred are expression libraries representing a display of the genetic set of a pathogen in recombinant form such as in vitro translation techniques, e.g. ribosomal display, or prokaryotic expression systems, e.g. bacterial surface expression libraries or which resemble specific physiological expression states of a given pathogen in a given physiological state, such as a proteome.

Ribosome display is an established method in recombinant DNA technology, which is applicable for each specific pathogen for the sake of the present invention (Schaffitzel et al, 1999). Bacterial surface display libraries will be represented by a recombinant library of a bacterial host displaying a (total) set of expressed peptide sequences of a given pathogen on e.g. a selected outer membrane protein at the bacterial host membrane (Georgiou et al., 1997). Apart from displaying peptide or protein sequences in an outer membrane protein, other bacterial display techniques, such as bacteriophage display technologies and expression via exported proteins are also preferred as bacterial surface expression library (Forrer et al., 1999; Rodi and Makowski, 1993; Georgiou et al., 1997).

The antigen preparation for the first round of screening in the method according to the present invention may be derived from any source containing antibodies to a given pathogen. Preferably, if a plasma pool is used as a source for the antibody preparation, a human plasma pool is selected which comprises donors which had experienced or are experiencing an infection with the given pathogen. Although such a selection of plasma or plasma pools is in principle standard technology in for example the production of hyperimmunoglobulin preparations, it was surprising that such technologies have these effects as especially shown for the preferred embodiments of the present invention.

Preferably the expression libraries are genomic expression libraries of a given pathogen, or alternatively m-RNA, libraries. It is preferred that these genomic or m-RNA libraries are complete genomic or m-RNA expression libraries which means that they contain at least once all possible proteins, peptides or peptide fragments of the given pathogen are expressable. Preferably the genomic expression libraries exhibit a redundancy of at least 2×, more preferred at least 5×, especially at least 10×.

Preferably, the method according to the present invention comprises screening at least a ribosomal display library, a bacterial surface display library and a proteome with the antibody preparation and identifying antigens which bind in at least two, preferably which bind to all, of said screenings to antibodies in said antibody preparation. Such antigens may then be regarded extremely suited as hyperimmunogenic antigens regardless of their way of expression. Preferably the at least two screenings should at least contain the proteome, since the proteome always represents the antigens as naturally expressed proteins including post-translational modifications, processing, etc. which are not obvious from the DNA sequence.

The method according to the present invention may be applied to any given pathogen. Therefore, preferred pathogens are selected from the group of bacterial, viral, fungal and protozoan pathogens. The method according to the present invention is also applicable to cancer, i.e. for the identification of tumor-associated antigens, and for the identification of allergens or antigens involved in auto-immune diseases. Of course, especially the recombinant methods are rather simple for pathogens having a small genome or a comparatively small number of expressed proteins (such as bacterial or viral pathogens) and are more complicated for complex (eukaryotic) organisms having large genomes. However, also such large genomic libraries of higher organism pathogens may well be analyzed with the method according to the present invention, at least in a faster and more reliable way than with known methods for identifying suitable antigens.

Preferred pathogens to be analyzed or which antigens are to be extracted, respectively, include human immunedeficiency virus (HIV), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), Rous sarcoma virus (RSV), Epstein-Barr virus (EBV), influenza virus (IV), rotavirus (RV), Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epidermidis), Chlamydia pneumoniae (C. pneumoniae), Chlamydia trachomatis (C. trachomatis), Mycobacterium tuberculosis (M. tuberculosis), Mycobacterium leprae (M. leprae), Streptococcus pneumoniae (S. pneumoniae), Streptococcus pyogenes (S. pyogenes), Streptococcus agalactiae (S. agalactiae), Enterococcus faecalis (E. faecalis), Bacillus anthracis (B. anthracis), Vibrio cholerae (V. cholerae), Borrelia burgdorferi (B. burgdorferi), Plasmodium sp., fungal diseases such as Pneumocystis carinii, Aspergillus sp., Cryptococcus sp., Candida albicans or parasitic infections such as ascariasis (Ascaris lumbricoides) and taeniasis (Taenia saginata). The method according to the present invention is most applicable for bacteria, worms or candida.

As a model organism for the present application Staphylococcus aureus has been chosen to demonstrate the applicability and efficacy of the method according to the present invention. Especially with respect to the examples it is clear that the invention is easily transferable to all potential pathogens, especially the ones listed above.

It was surprising that the method according to the present invention allows an efficient and fast biological screening of a given pathogen, especially in view of the fact that only a small fraction of a patient's antibody repertoire is directed to a given pathogen, even in a state where this pathogen is effectively defeated. It has been discovered within the course of the present invention, especially during performance of the S. aureus example that only 1-2% of the antibody repertoire of a patient having high titers against S. aureus are indeed antibodies directed against S. aureus. Moreover, over 70% of this specific 1% portion is directed against non-protein antigens, such as teichoic acid, so that only a total of 0.1% or less of the antibodies are directed to proteinaceous antigens.

One of the advantages of using recombinant expression libraries, especially ribosome display libraries and bacterial surface display libraries, is that the identified hyperimmune serum-reactive antigens may be instantly produced by expression of the coding sequences of the screened and selected clones expressing the hyperimmune serum-reactive antigens without further recombinant DNA technology or cloning steps necessary.

The hyperimmune serum-reactive antigens obtainable by the method according to the present invention may therefore be immediately finished to a pharmaceutical preparation, preferably by addition of a pharmaceutically acceptable carrier and/or excipient, immediately after its production (in the course of the second selection step), e.g. by expression from the expression library platform.

Preferably, the pharmaceutical preparation containing the hyperimmune serum-reactive antigen is a vaccine for preventing or treating an infection with the specific pathogen for which the antigens have been selected.

The pharmaceutical preparation may contain any suitable auxiliary substances, such as buffer substances, stabilisers or further active ingredients, especially ingredients known in connection of vaccine production.

A preferable carrier/or excipient for the hyperimmune serum-reactive antigens according to the present invention is a immunostimulatory compound for further stimulating the immune response to the given hyperimmune serum-reactive antigen. Preferably the immunostimulatory compound in the pharmaceutical preparation according to the present invention is selected from the group of polycationic substances, especially polycationic peptides, immunostimulatory deoxynucleotides, alumn, Freund's complete adjuvans, Freund's incomplete adjuvans, neuroactive compounds, especially human growth hormone, or combinations thereof.

The polycationic compound(s) to be used according to the present invention may be any polycationic compound which shows the characteristic effects according to the WO 97/30721. Preferred polycationic compounds are selected from basic polypeptides, organic polycations, basic polyamino acids or mixtures thereof. These polyamino acids should have a chain length of at least 4 amino acid residues (see: Tuftsin as described in Goldman et al. (1983)). Especially preferred are substances like polylysine, polyarginine and polypeptides containing more than 20%, especially more than 50% of basic amino acids in a range of more than 8, especially more than 20, amino acid residues or mixtures thereof. Other preferred polycations and their pharmaceutical compositions are described in WO 97/30721 (e.g. polyethyleneimine) and WO 99/38528. Preferably these polypeptides contain between 20 and 500 amino acid residues, especially between 30 and 200 residues.

These polycationic compounds may be produced chemically or recombinantly or may be derived from natural sources.

Cationic (poly)peptides may also be anti-microbial with properties as reviewed in Ganz et al, 1999; Hancock, 1999. These (poly)peptides may be of prokaryotic or animal or plant origin or may be produced chemically or recombinantly (Andreu et al., 1998; Ganz et al., 1999; Simmaco et al., 1998). Peptides may also belong to the class of defensins (Ganz, 1999; Ganz et al., 1999). Sequences of such peptides can be, for example, found in the Antimicrobial Sequences Database on the world wide web at: bbcm.univ.trieste.it/.about.tossi/pag2.html

Such host defence peptides or defensives are also a preferred form of the polycationic polymer according to the present invention. Generally, a compound allowing as an end product activation (or down-regulation) of the adaptive immune system, preferably mediated by APCs (including dendritic cells) is used as polycationic polymer.

Especially preferred for use as polycationic substance in the present invention are cathelicidin derived antimicrobial peptides or derivatives thereof (International patent application PCT/EP01/09529, incorporated herein by reference), especially antimicrobial peptides derived from mammal cathelicidin, preferably from human, bovine or mouse.

Polycationic compounds derived from natural sources include HIV-REV or HIV-TAT (derived cationic peptides, antennapedia peptides, chitosan or other derivatives of chitin) or other peptides derived from these peptides or proteins by biochemical or recombinant production. Other preferred polycationic compounds are cathelin or related or derived substances from cathelin. For example, mouse cathelin is a peptide which has the amino acid sequence NH₂-RLAGLLRKGGEKIGEKLKKIGQIKIKNFFQKLVPQPE-COOH (SEQ ID NO:599). Related or derived cathelin substances contain the whole or parts of the cathelin sequence with at least 15-20 amino acid residues. Derivations may include the substitution or modification of the natural amino acids by amino acids which are not among the 20 standard amino acids. Moreover, further cationic residues may be introduced into such cathelin molecules. These catheline molecules are preferred to be combined with the antigen. These cathelin molecules surprisingly have turned out to be also effective as an adjuvant for an antigen without the addition of further adjuvants. It is therefore possible to use such cathelin molecules as efficient adjuvants in vaccine formulations with or without further immunactivating substances.

Another preferred polycationic substance to be used according to the present invention is a synthetic peptide containing at least 2 KLK-motifs separated by a linker of 3 to 7 hydrophobic amino acids (International patent application PCT/EP01/12041, incorporated herein by reference).

Immunostimulatory deoxynucleotides are e.g. neutral or artificial CpG containing DNA, short stretches of DNA derived from non-vertebrates or in form of short oligonucleotides (ODNs) containing non-methylated cytosine-guanine di-nucleotides (CpG) in a certain base context (e.g. Krieg et al., 1995) but also inosine containing ODNs (I-ODNs) as described in WO 01/93905.

Neuroactive compounds, e.g. combined with polycationic substances are described in WO 01/24822.

According to a preferred embodiment the individual antibody preparation for the second round of screening are derived from patients with have suffered from an acute infection with the given pathogen, especially from patients who show an antibody titer to the given pathogen above a certain minimum level, for example an antibody titer being higher than 80 percentile, preferably higher than 90 percentile, especially higher than 95 percentile of the human (patient or carrier) sera tested. Using such high titer individual antibody preparations in the second screening round allows a very selective identification of the hyperimmune serum-reactive antigens to the given pathogen.

It is important that the second screening with the individual antibody preparations (which may also be the selected serum) allows a selective identification of the hyperimmune serum-reactive antigens from all the promising candidates from the first round. Therefore, preferably at least 10 individual antibody preparations (i.e. antibody preparations (e.g. sera) from at least 10 different individuals having suffered from an infection to the chosen pathogen) should be used in identifying these antigens in the second screening round. Of course, it is possible to use also less than 10 individual preparations, however, selectivity of the step may not be optimal with a low number of individual antibody preparations. On the other hand, if a given hyperimmune serum-reactive antigen (or an antigenic fragment thereof) is recognized in at least 10 individual antibody preparations, preferably at least 30, especially at least 50 individual antibody preparations, identification of hyperimmune serum-reactive antigen is also selective enough for a proper identification. Hyperimmune serum-reactivity may of course be tested with as many individual preparations as possible (e.g. with more than 100 or even with more than 1000).

Therefore, the relevant portion of the hyperimmune serum-reactive antibody preparation according to the method of the present invention should preferably be at least 10, more preferred at least 30, especially at least 50 individual antibody preparations. Alternatively (or in combination) hyperimmune serum-reactive antigen may preferably be also identified with at least 20%, preferably at least 30%, especially at least 40% of all individual antibody preparations used in the second screening round.

According to a preferred embodiment of the present invention, the sera from which the individual antibody preparations for the second round of screening are prepared (or which are used as antibody preparations), are selected by their titer against the specific pathogen (e.g. against a preparation of this pathogen, such as a lysate, cell wall components and recombinant proteins). Preferably, some are selected with a total IgA titer above 4000 U, especially above 6000 U, and/or an IgG titer above 10 000 U, especially above 12 000 U (U=units, calculated from the OD_(405 nm) reading at a given dilution) when whole organism (total lysate or whole cells) is used as antigen in ELISA. Individual proteins with Ig titers of above 800-1000 U are specifically preferred for selecting the hyperimmune serum-reactive antigens according to the present invention only for total titer. The statement for individual proteins can be derived from FIG. 9.

According to the demonstration example which is also a preferred embodiment of the present invention the given pathogen is a Staphylococcus pathogen, especially Staphylococcus aureus and Staphylococcus epidermidis. Staphylococci are opportunistic pathogens which can cause illnesses which range from minor infections to life threatening diseases. Of the large number of Staphylococci at least 3 are commonly associated with human disease: S. aureus, S. epidermidis and rarely S. saprophyticus (Crossley and Archer, 1997). S. aureus has been used within the course of the present invention as an illustrative example of the way the present invention functions. Besides that, it is also an important organism with respect to its severe pathogenic impacts on humans. Staphylococcal infections are imposing an increasing threat in hospitals worldwide. The appearance and disease causing capacity of Staphylococci are related to the wide-spread use of antibiotics which induced and continue to induce multi-drug resistance. For that reason medical treatment against Staphylococcal infections cannot rely only on antibiotics anymore. Therefore, a tactic change in the treatment of these diseases is desperately needed which aims to prevent infections. Inducing high affinity antibodies of the opsonic and neutralizing type by vaccination helps the innate immune system to eliminate bacteria and toxins. This makes the method according to the present invention an optimal tool for the identification of staphylococcal antigenic proteins.

Every human being is colonized with S. epidermidis. The normal habitats of S. epidermidis are the skin and the mucous membrane. The major habitats of the most pathogenic species, S. aureus, are the anterior nares and perineum. Some individuals become permanent S. aureus carriers, often with the same strain. The carrier stage is clinically relevant because carriers undergoing surgery have more infections than noncarriers. Generally, the established flora of the nose prevents acquisition of new strains. However, colonization with other strains may occur when antibiotic treatment is given that leads to elimination of the susceptible carrier strain. Because this situation occurs in the hospitals, patients may become colonized with resistant nosocomial Staphylococci. These bacteria have an innate adaptability which is complemented by the widespread and sometimes inappropriate use of antimicrobial agents. Therefore hospitals provide a fertile environment for drug resistance to develop (close contact among sick patients, extensive use of antimicrobials, nosocomial infections). Both S. aureus and S. epidermidis have become resistant to many commonly used antibiotics, most importantly to methicillin (MRSA) and vancomycin (VISA). Drug resistance is an increasingly important public health concern, and soon many infections caused by staphylococci may be untreatable by antibiotics. In addition to its adverse effect on public health, antimicrobial resistance contributes to higher health care costs, since treating resistant infections often requires the use of more toxic and more expensive drugs, and can result in longer hospital stays for infected patients.

Moreover, even with the help of effective antibiotics, the most serious staphylococcal infections have 30-50% mortality.

Staphylococci become potentially pathogenic as soon as the natural balance between microorganisms and the immune system gets disturbed, when natural barriers (skin, mucous membrane) are breached. The coagulase-positive S. aureus is the most pathogenic staphylococcal species, feared by surgeons for a long time. Most frequently it causes surgical wound infections, and induces the formation of abscesses. This local infection might become systemic, causing bacteraemia and sepsis. Especially after viral infections and in elderly, it can cause severe pneumonia. S. aureus is also a frequent cause of infections related to medical devices, such as intravascular and percutan catheters (endocarditis, sepsis, peritonitis), prosthetic devices (septic arthritis, osteomyelitis). S. epidermidis causes diseases mostly related to the presence of foreign body and the use of devices, such as catheter related infections, cerebrospinal fluid shunt infections, peritonitis in dialysed patients (mainly CAPD), endocarditis in individuals with prosthetic valves. This is exemplified in immunocompromised individuals such as oncology patients and premature neonates in whom coagulase-negative staphylococcal infections frequently occur in association with the use of intravascular device. The increase in incidence is related to the increased used of these devices and increasing number of immuno-compromised patients.

Much less is known about S. saprophyticus, another coagulase-negative staphylococci, which causes acute urinary tract infection in previously healthy people. With a few exceptions these are women aged 16-25 years.

The pathogenesis of staphylococci is multifactorial. In order to initiate infection the pathogen has to gain access to the cells and tissues of the host, that is adhere. S. aureus expresses-surface proteins that promote attachment to the host proteins such as laminin, fibronectin, elastin, vitronectin, fibrinogen and many other molecules that form part of the extracellular matrix (extracellular matrix binding proteins, ECMBP). S. epidermidis is equipped with cell surface molecules which promote adherence to foreign material and through that mechanism establish infection in the host. The other powerful weapons staphylococci use are the secreted products, such as enterotoxins, exotoxins, and tissue damaging enzymes. The toxins kill or misguide immune cells which are important in the host defence. The several different types of toxins are responsible for most of the symptoms during infections.

Host defence against S. aureus relies mainly on innate immunological mechanisms. The skin and mucous membranes are formidable barriers against invasion by Staphylococci. However, once the skin or the mucous membranes are breached (wounds, percutan catheters, etc), the first line of nonadaptive cellular defence begins its co-ordinate action through complement and phagocytes, especially the polymorphonuclear leukocytes (PMNs). These cells can be regarded as the cornerstones in eliminating invading bacteria. As Staphylococci are primarily extracellular pathogens; the major anti-staphylococcal adaptive response comes from the humoral arm of the immune system, and is mediated through three major mechanisms: promotion of opsonization, toxin neutralisation, and inhibition of adherence. It is believed that opsonization is especially important, because of its requirement for an effective phagocytosis. For efficient opsonization the microbial surface has to be coated with antibodies and complement factors for recognition by PMNs through receptors to the Fc fragment of the IgG molecule or to activated C3b. After opsonization, staphylococci are phagocytosed and killed. Moreover, S. aureus can attach to endothelial cells, and be internalised by a phagocytosis-like process. Antibodies bound to specific antigens on the cell surface of bacteria serve as ligands for the attachment to PMNs and promote phagocytosis. The very same antibodies bound to the adhesins and other cell surface proteins are expected to neutralize adhesion and prevent colonization.

There is little clinical evidence that cell mediated immunity has a significant contribution in the defence against Staphylococci, yet one has to admit that the question is not adequately addressed. It is known, however, that Staphylococcus aureus utilizes an extensive array of molecular countermeasures to manipulate the defensive microenvironment of the infected host by secreting polypeptides referred to as superantigens, which target the multireceptor communication between T-cells and antigen-presenting cells that is fundamental to initiating pathogen-specific immune clearance. Superantigens play a critical role in toxic shock syndrome and food poisoning, yet their function in routine infections is not well understood. Moreover, one cannot expect a long lasting antibody (memory) response without the involvement of T-cells. It is also known that the majority of the anti-staphylococcal antibodies are against T-cell independent antigens (capsular polysaccharides, lipoteichoic acid, peptidoglycan) without a memory function. The T-cell dependent proteinaceous antigens can elicit long-term protective antibody responses. These staphylococcal proteins and peptides have not yet been determined.

For all these above mentioned reasons, a tactic change on the war field against staphylococcal infections is badly needed. One way of combating infections is preventing them by active immunisation. Vaccine development against S. aureus has been initiated by several research groups and national institutions worldwide, but there is no effective vaccine approved so far. It has been shown that an antibody deficiency state contributes to staphylococcal persistence, suggesting that anti-staphylococcal antibodies are important in host defence. Antibodies—added as passive immunisation or induced by active vaccination—directed towards surface components could both prevent bacterial adherence, neutralize toxins and promote phagocytosis. A vaccine based on fibronectin binding protein induces protective immunity against mastitis in cattle and suggest that this approach is likely to work in humans (refs). Taking all this together it is suggestive that an effective vaccine should be composed of proteins or polypeptides, which are expressed by all strains and are able to induce high affinity, abundant antibodies against cell surface components of S. aureus. The antibodies should be IgG1 and/or IgG3 for opsonization, and any IgG subtype and IgA for neutralisation of adherence and toxin action. A chemically defined vaccine must be definitely superior compared to a whole cell vaccine (attenuated or killed), since components of S. aureus which paralyze TH cells (superantigens) or inhibit opsonization (protein A) can be eliminated, and the individual proteins inducing protective antibodies can be selected. Identification of the relevant antigens help to generate effective passive immunisation (humanised monoclonal antibody therapy), which can replace human immunoglobulin administration with all its dangerous side-effects. Neonatal staphylococcal infections, severe septicemia and other life-threatening acute conditions are the primary target of passive immunisation. An effective vaccine offers great potential for patients facing elective surgery in general, and those receiving endovascular devices, in particular. Moreover, patients suffering from chronic diseases which decrease immune responses or undergoing continuous ambulatory peritoneal dialysis are likely to benefit from such a vaccine.

For the illustrative example concerning Staphylococcus aureus three different approaches have been employed in parallel. All three of these methods are based on the interaction of Staphylococcus proteins or peptides with the antibodies present in human sera with the method according to the present invention. This interaction relies on the recognition of epitopes within the proteins which can be short peptides (linear epitopes) or polypeptide domains (structural epitopes). The antigenic proteins are identified by the different methods using pools of pre-selected sera and—in the second screening round—by individual selected sera.

Following the high throughput screening, the selected antigenic proteins are expressed as recombinant proteins or in vitro translated products (in case it can not be expressed in prokaryotic expression systems), and tested in a series of ELISA and Western blotting assays for the assessment of immunogenicity with a large human serum collection (>100 uninfected, >50 patients sera). The preferred antigens are located on the cell surface or secreted, that is accessible extracellularly. Antibodies against the cell wall proteins (such as the Extracellular matrix binding proteins) are expected to serve double purposes: to inhibit adhesion and promote phagocytosis. The antibodies against the secreted proteins are beneficial in toxin neutralisation. It is also known that bacteria communicate with each other through secreted proteins. Neutralizing antibodies against these proteins will interrupt growth promoting cross-talk between or within staphylococcal species. Bioinformatics (signal sequences, cell wall localisation signals, transmembrane domains) proved to be very useful in assessing cell surface localisation or secretion. The experimental approach includes the isolation of antibodies with the corresponding epitopes and proteins from human serum, and use them as reagents in the following assays: cell surface staining of staphylococci grown under different conditions (FACS, microscopy), determination of neutralizing capacity (toxin, adherence), and promotion of opsonization and phagocytosis (in vitro phagocytosis assay).

The recognition of linear epitopes by antibodies can be based on sequences as short as 4-5 aa. Of course it does not necessarily mean that these short peptides are capable of inducing the given antibody. in vivo. For that reason the defined epitopes, polypeptides and proteins may further be tested in animals (mainly in mice) for their capacity to induce antibodies against the selected proteins in vivo. The antigens with the proven capability to induce antibodies will be tested in animal models for the ability to prevent infections.

The antibodies produced against Staphylococci by the human immune system and present in human sera are indicative of the in vivo expression of the antigenic proteins and their immunogenicity.

Accordingly, novel hyperimmune serum-reactive antigens from Staphylococcus aureus or Staphylococcus epidermidis have been made available by the method according to the present invention. According to another aspect of the present invention the invention relates to a hyperimmune serum-reactive antigen selected from the group consisting of the sequences listed in any one of Tables 2a, 2b, 2c, 2d, 3, 4 and 5, especially selected from the group consisting of Seq. ID No. 56, 57, 59, 60, 67, 70, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 85, 87, 88, 89, 90, 92, 95, 96, 97, 99, 100, 101, 102, 103, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 126, 128, 132, 134, 138, 140, 142, 151, 152, 154, 155 and hyperimmune fragments thereof. Accordingly, the present invention also relates to a hyperimmune serum-reactive antigen obtainable by the method according to the present invention and being selected from the group consisting of the sequences listed in any one of Tables 2a, 2b, 2c, 2d, 3, 4 and 5, especially selected from the group consisting of Seq. ID No. 56, 57, 59, 60, 67, 70, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 85, 87, 88, 89, 90, 92, 95, 96, 97, 99, 100, 101, 102, 103, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 126, 128, 132, 134, 138, 140, 142, 151, 152, 154, 155 and hyperimmune fragments thereof.

Antigens from Staphylococcus aureus and Staphylococcus epidermidis have been extracted by the method according to the present invention which may be used in the manufacture of a pharmaceutical preparation, especially for the manufacture of a vaccine against Staphylococcus aureus and Staphylococcus epidermidis infections. Examples of such hyperimmune serum-reactive antigens of Staphylococcus aureus and Staphylococcus epidermidis to be used in a pharmaceutical preparation are selected from the group consisting of the sequences listed in any one of Tables 2a, 2b, 2c, 2d, 3, 4 and 5, especially selected from the group consisting of Seq. ID No. 55, 56, 57, 58, 59, 60, 62, 66, 67, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87, 88, 89, 90, 92, 94, 95, 96, 97, 99, 100, 101, 102, 103, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 126, 128, 130, 132, 134, 138, 140, 142, 151, 152, 154, 155, 158 and hyperimmune fragments thereof for the manufacture of a pharmaceutical preparation, especially for the manufacture of a vaccine against Staphylococcus aureus and Staphylococcus epidermidis infections.

A hyperimmune fragment is defined as a fragment of the identified antigen which is for itself antigenic or may be made antigenic when provided as a hapten. Therefore, also antigen or antigenic fragments showing one or (for longer fragments) only a few amino acid exchanges are enabled with the present invention, provided that the antigenic capacities of such fragments with amino acid exchanges are not severely deteriorated on the exchange(s). i.e. suited for eliciting an appropriate immune response in a individual vaccinated with this antigen and identified by individual antibody preparations from individual sera.

Preferred examples of such hyperimmune fragments of a hyperimmune serum-reactive antigen are selected from the group consisting of peptides comprising the amino acid sequences of column “predicted immunogenic aa”, “Location of identified immunogenic region” and “Serum reactivity with relevant region” of Tables 2a, 2b, 2c and 2d and the amino acid sequences of column “Putative antigenic surface areas” of Table 4 and 5, especially peptides comprising amino acid No. aa 12-29, 34-40, 63-71, 101-110, 114-122, 130-138, 140-195, 197-209, 215-229, 239-253, 255-274 and 39-94 of Seq. ID No. 55,

aa 5-39, 111-117, 125-132, 134-141, 167-191, 196-202, 214-232, 236-241, 244-249, 292-297, 319-328, 336-341, 365-380, 385-391, 407-416, 420-429, 435-441, 452-461, 477-488, 491-498, 518-532, 545-556, 569-576, 581-587, 595-602, 604-609, 617-640, 643-651, 702-715, 723-731, 786-793, 805-811, 826-839, 874-889, 37-49, 63-77 and 274-334, of Seq. ID No. 56, aa 28-55, 82-100, 105-111, 125-131, 137-143, 1-49, of Seq. ID No. 57, aa 33-43, 45-51, 57-63, 65-72, 80-96, 99-110, 123-129, 161-171, 173-179, 185-191, 193-200, 208-224, 227-246, 252-258, 294-308, 321-329, 344-352, 691-707, 358-411 and 588-606, of Seq. ID No. 58, aa 16-38, 71-77, 87-94, 105-112, 124-144, 158-164, 169-177, 180-186, 194-204, 221-228, 236-245, 250-267, 336-343, 363-378, 385-394, 406-412, 423-440, 443-449, 401-494, of Seq. ID No. 59, aa 18-23, 42-55, 69-77, 85-98, 129-136, 182-188, 214-220, 229-235, 242-248, 251-258, 281-292, 309-316, 333-343, 348-354, 361-367, 393-407, 441-447, 481-488, 493-505, 510-515, 517-527, 530-535, 540-549, 564-583, 593-599, 608-621, 636-645, 656-670, 674-687, 697-708, 726-734, 755-760, 765-772, 785-792, 798-815, 819-824, 826-838, 846-852, 889-904, 907-913, 932-939, 956-964, 982-1000, 1008-1015, 1017-1024, 1028-1034, 1059-1065, 1078-1084, 1122-1129, 1134-1143, 1180-1186, 1188-1194, 1205-1215, 1224-1230, 1276-1283, 1333-1339, 1377-1382, 1415-1421, 1448-1459, 1467-1472, 1537-1545, 1556-1566, 1647-1654, 1666-1675, 1683-1689, 1722-1737, 1740-1754, 1756-1762, 1764-1773, 1775-1783, 1800-1809, 1811-1819, 1839-1851, 1859-1866, 1876-1882, 1930-1939, 1947-1954, 1978-1985, 1999-2007, 2015-2029, 2080-2086, 2094-2100, 2112-2118, 2196-2205, 2232-2243, 198-258, 646-727 and 2104-2206, of Seq. ID No. 60, aa 10-29, 46-56, 63-74, 83-105, 107-114, 138-145, 170-184, 186-193, 216-221, 242-248, 277-289, 303-311, 346-360, 379-389, 422-428, 446-453, 459-469, 479-489, 496-501, 83-156, of Seq. ID No. 62, aa 14-22, 32-40, 52-58, 61-77, 81-93, 111-117, 124-138, 151-190, 193-214, 224-244, 253-277, 287-295, 307-324, 326-332, 348-355, 357-362, 384-394, 397-434, 437-460, 489-496, 503-510, 516-522, 528-539, 541-547, 552-558, 563-573, 589-595, 602-624, 626-632, 651-667, 673-689, 694-706, 712-739, 756-790, 403-462, of Seq. ID No. 66, aa 49-56, 62-68, 83-89, 92-98, 10.9-115, 124-131, 142-159, 161-167, 169-175, 177-188, 196-224, 230-243, 246-252, 34-46, of Seq. ID No. 67, aa 11-20, 26-47, 69-75, 84-92, 102-109, 119-136, 139-147, 160-170, 178-185, 190-196, 208-215, 225-233, 245-250, 265-272, 277-284, 300-306, 346-357, 373-379, 384-390, 429-435, 471-481, 502-507, 536-561, 663-688, 791-816, 905-910, 919-933, 977-985, 1001-1010, 1052-1057, 1070-1077, 1082-1087, 1094-1112, 493-587, 633-715 and 704-760, of Seq. ID No. 70, aa 6-20, 53-63, 83-90, 135-146, 195-208, 244-259, 263-314, 319-327, 337-349, 353-362, 365-374, 380-390, 397-405, 407-415, 208-287 and 286-314, of Seq. ID No. 71, aa 10-26, 31-43, 46-58, 61-66, 69-79, 85-92, 100-115, 120-126, 128-135, 149-155, 167-173, 178-187, 189-196, 202-222, 225-231, 233-240, 245-251, 257-263, 271-292, 314-322, 325-334, 339-345, 59-74, of Seq. ID No. 72, aa 4-9, 15-26, 65-76, 108-115, 119-128, 144-153, 38-52 and 66-114, of Seq. ID No. 73, aa 5-22, 42-50, 74-81, 139-145, 167-178, 220-230, 246-253, 255-264, 137-237 and 250-267, of Seq. ID No. 74, aa 10-26, 31-44, 60-66, 99-104, 146-153, 163-169, 197-205, 216-223, 226-238, 241-258, 271-280, 295-315, 346-351, 371-385, 396-407, 440-446, 452-457, 460-466, 492-510, 537-543, 546-551, 565-582, 590-595, 635-650, 672-678, 686-701, 705-712, 714-721, 725-731, 762-768, 800-805, 672-727, of Seq. ID No. 75, aa 5-32, 35-48, 55-76, of Seq. ID No. 76, aa 7-35, 54-59, 247-261, 263-272, 302-320, 330-339, 368-374, 382-411, 126-143 and 168-186, of Seq. ID No. 77, aa 5-24, 88-94, 102-113, 132-143, 163-173, 216-224, 254-269, 273-278, 305-313, 321-327, 334-341, 31-61 and 58-74, of Seq. ID No. 78, aa 16-24, 32-39, 43-49, 64-71, 93-99, 126-141, 144-156, 210-218, 226-233, 265-273, 276-284, 158-220, of Seq. ID No. 79, aa 49-72, 76-83, 95-105, 135-146, 148-164, 183-205, 57-128, of Seq. ID No. 80, aa 6-15, 22-32, 58-73, 82-88, 97-109, 120-131, 134-140, 151-163, 179-185, 219-230, 242-255, 271-277, 288-293, 305-319, 345-356, 368-381, 397-406, 408-420, 427-437, 448-454, 473-482, 498-505, 529-535, 550-563, 573-580, 582-590, 600-605, 618-627, 677-685, 718-725, 729-735, 744-759, 773-784, 789-794, 820-837, 902-908, 916-921, 929-935, 949-955, 1001-1008, 1026-1032, 1074-1083, 1088-1094, 1108-1117, 1137-1142, 1159-1177, 1183-1194, 1214-1220, 1236-1252, 1261-1269, 1289-1294, 1311-1329, 1336-1341, 1406-1413, 1419-1432, 1437-1457, 1464-1503, 1519-1525, 1531-1537, 1539-1557, 1560-1567, 1611-1618, 1620-1629, 1697-1704, 1712-1719, 1726-1736, 1781-1786, 1797-1817, 1848-1854, 1879-1890, 1919-1925, 1946-1953, 1974-1979, 5 to 134, of Seq. ID No. 81, aa 6-33, 40-46, 51-59, 61-77, 84-104, 112-118, 124-187, 194-248, 252-296, 308-325, 327-361, 367-393, 396-437, 452-479, 484-520, 535-545, 558-574, 582-614, 627-633, 656-663, 671-678, 698-704, 713-722, 725-742, 744-755, 770-784, 786-800, 816-822, 827-837, 483-511, of Seq. ID No. 82, aa 4-19, 57-70, 79-88, 126-132, 144-159, 161-167, 180-198, 200-212, 233-240, 248-255, 276-286, 298-304, 309-323, 332-346, 357-366, 374-391, 394-406, 450-456, 466-473, 479-487, 498-505, 507-519, 521-530, 532-540, 555-565, 571-581, 600-611, 619-625, 634-642, 650-656, 658-665, 676-682, 690-699, 724-733, 740-771, 774-784, 791-797, 808-815, 821-828, 832-838, 876-881, 893-906, 922-929, 938-943, 948-953, 969-976, 1002-1008, 1015-1035, 1056-1069, 1105-1116, 1124-1135, 1144-1151, 1173-1181, 1186-1191, 1206-1215, 1225-1230, 1235-1242, 6-66, 65-124 and 590-604, of Seq. ID No. 83, aa 5-32, 66-72, 87-98, 104-112, 116-124, 128-137, 162-168, 174-183, 248-254, 261-266, 289-303, 312-331, 174-249, of Seq. ID No. 84, aa 4-21, 28-40, 45-52, 59-71, 92-107, 123-137, 159-174, 190-202, 220-229, 232-241, 282-296, 302-308, 312-331, 21-118, of Seq. ID No. 85, aa 9-28, 43-48, 56-75, 109-126, 128-141, 143-162, 164-195, 197-216, 234-242, 244-251, 168-181, of Seq. ID No. 87, aa 4-10, 20-42, 50-86, 88-98, 102-171, 176-182, 189-221, 223-244, 246-268, 276-284, 296-329, 112-188, of Seq. ID No. 88, aa 4-9, 13-24, 26-34, 37-43, 45-51, 59-73, 90-96, 99-113, 160-173, 178-184, 218-228, 233-238, 255-262, 45-105, 103-166 and 66-153, of Seq. ID No. 89, aa 13-27, 42-63, 107-191, 198-215, 218-225, 233-250, 474-367, of Seq. ID No. 90, aa 26-53, 95-123, 164-176, 189-199, 8-48, of Seq. ID No. 92, aa 7-13, 15-23, 26-33, 68-81, 84-90, 106-117, 129-137, 140-159, 165-172, 177-230, 234-240, 258-278, 295-319, 22-56, 23-99, 97-115, 233-250 and 245-265, of Seq. ID No. 94, aa 13-36, 40-49, 111-118, 134-140, 159-164, 173-183, 208-220, 232-241, 245-254, 262-271, 280-286, 295-301, 303-310, 319-324, 332-339, 1-85, 54-121 and 103-185, of Seq. ID No. 95, aa 39-44, 46-80, 92-98, 105-113, 118-123, 133-165, 176-208, 226-238, 240-255, 279-285, 298-330, 338-345, 350-357, 365-372, 397-402, 409-415, 465-473, 488-515, 517-535, 542-550, 554-590, 593-601, 603-620, 627-653, 660-665, 674-687, 698-718, 726-739, 386-402, of Seq. ID No. 96, aa 5-32, 34-49, 1-43, of Seq. ID No. 97, aa 10-27, 37-56, 64-99, 106-119, 121-136, 139-145, 148-178, 190-216, 225-249, 251-276, 292-297, 312-321, 332-399, 403-458, 183-200, of Seq. ID No. 99, aa 5-12, 15-20, 43-49, 94-106, 110-116, 119-128, 153-163, 175-180, 185-191, 198-209, 244-252, 254-264, 266-273, 280-288, 290-297, 63-126, of Seq. ID No. 100, aa 5-44, 47-55, 62-68, 70-78, 93-100, 128-151, 166-171, 176-308, 1-59, of Seq. ID No. 101, aa 18-28, 36-49, 56-62, 67-84, 86-95, 102-153, 180-195, 198-218, 254-280, 284-296, 301-325, 327-348, 353-390, 397-402, 407-414, 431-455, 328-394, of Seq. ID No. 102, aa 7-37, 56-71, 74-150, 155-162, 183-203, 211-222, 224-234, 242-272, 77-128, of Seq. ID No. 103, aa 34-58, 63-69, 74-86, 92-101, 130-138, 142-150, 158-191, 199-207, 210-221, 234-249, 252-271, 5-48, of Seq. ID No. 104, aa 12-36, 43-50, 58-65, 73-78, 80-87, 108-139, 147-153, 159-172, 190-203, 211-216, 224-232, 234-246, 256-261, 273-279, 286-293, 299-306, 340-346, 354-366, 167-181, of Seq. ID No. 106, aa 61-75, 82-87, 97-104, 113-123, 128-133, 203-216, 224-229, 236-246, 251-258, 271-286, 288-294, 301-310, 316-329, 337-346, 348-371, 394-406, 418-435, 440-452 of Seq. ID No. 112, aa 30-37, 44-55, 83-91, 101-118, 121-128, 136-149, 175-183, 185-193, 206-212, 222-229, 235-242 of Seq. ID No. 114, aa 28-38, 76-91, 102-109, 118-141, 146-153, 155-161, 165-179, 186-202, 215-221, 234-249, 262-269, 276-282, 289-302, 306-314, 321-326, 338-345, 360-369, 385-391 of Seq. ID No. 116, aa 9-33, 56-62, 75-84, 99-105, 122-127, 163-180, 186-192, 206-228, 233-240, 254-262, 275-283, 289-296, 322-330, 348-355, 416-424, 426-438, 441-452, 484-491, 522-528, 541-549, 563-569, 578-584, 624-641, 527-544, of Seq. ID No. 142, aa 37-42, 57-62, 121-135, 139-145, 183-190, 204-212, 220-227, 242-248, 278-288, 295-30, 304-309, 335-341, 396-404, 412-433, 443-449, 497-503, 505-513, 539-545, 552-558, 601-617, 629-649, 702-711, 736-745, 793-804, 814-829, 843-858, 864-885, 889-895, 905-913, 919-929, 937-943, 957-965, 970-986, 990-1030, 1038-1049, 1063-1072, 1080-1091, 1093-1116, 1126-1136, 1145-1157, 1163-1171, 1177-1183, 1189-1196, 1211-1218, 1225-1235, 1242-1256, 1261-1269, 624-684, of Seq. ID No. 151, aa 8-23, 31-38, 42-49, 61-77, 83-90, 99-108, 110-119, 140-147, 149-155, 159-171, 180-185, 189-209, 228-234, 245-262, 264-275, 280-302, 304-330, 343-360, 391-409, 432-437, 454-463, 467-474, 478-485, 515-528, 532-539, 553-567, 569-581, 586-592, 605-612, 627-635, 639-656, 671-682, 700-714, 731-747, 754-770, 775-791, 797-834, 838-848, 872-891, 927-933, 935-942, 948-968, 976-986, 1000-1007, 1029-1037, 630-700, of Seq. ID No. 152, aa 17-25, 27-55, 84-90, 95-101, 115-121, 55-101, of Seq. ID No. 154, aa 13-28, 40-46, 69-75, 86-92, 114-120, 126-137, 155-172, 182-193, 199-206, 213-221, 232-238, 243-253, 270-276, 284-290, 22-100, of Seq. ID No. 155 and aa 7-19, 46-57, 85-91, 110-117, 125-133, 140-149, 156-163, 198-204, 236-251, 269-275, 283-290, 318-323, 347-363, 9-42 and 158-174, of Seq. ID No. 158, aa 7-14, 21-30, 34-50, 52-63, 65-72, 77-84, 109-124, 129-152, 158-163, 175-190, 193-216, 219-234 of Seq. ID. No. 168, aa 5-24, 38-44, 100-106, 118-130, 144-154, 204-210, 218-223, 228-243, 257-264, 266-286, 292-299 of Seq. ID. No. 174, aa 29-44, 74-83, 105-113, 119-125, 130-148, 155-175, 182-190, 198-211, 238-245 of Seq. ID. No. 176, and fragments comprising at least 6, preferably more than 8, especially more than 10 aa of said sequences. All these fragments individually and each independently form a preferred selected aspect of the present invention.

Especially suited helper epitopes may also be derived from these antigens. Especially preferred helper epitopes are peptides comprising fragments selected from the peptides mentioned in column “Putative antigenic surface areas” in Tables 4 and 5 and from the group of aa 6-40, 583-598, 620-646 and 871-896 of Seq. ID. No. 56, aa 24-53 of Seq. ID. No. 70, aa 240-260 of Seq. ID. No. 74, aa 1660-1682 and 1746-1790 of Seq. ID. No. 81, aa 1-29, 680-709, and 878-902 of Seq. ID. No. 83, aa 96-136 of Seq. ID. No. 89, aa 1-29, 226-269 and 275-326 of Seq. ID. No. 94, aa 23-47 and 107-156 of Seq. ID. No. 114 and aa 24-53 of Seq. ID. No. 142 and fragments thereof being T-cell epitopes.

According to another aspect, the present invention relates to a vaccine comprising such a hyperimmune serum-reactive antigen or a fragment thereof as identified above for Staphylococcus aureus and Staphylococcus epidermidis. Such a vaccine may comprise one or more antigens against S. aureus or S. epidermidis. Optionally, such S. aureus or S. epidermidis antigens may also be combined with antigens against other pathogens in a combination vaccine. Preferably this vaccine further comprises an immunostimulatory substance, preferably selected from the group comprising polycationic polymers, especially polycationic peptides, immunostimulatory deoxynucleotides (ODNs), neuroactive compounds, especially human growth hormone, alumn, Freund's complete or incomplete adjuvans or combinations thereof. Such a vaccine may also comprise the antigen displayed on a surface display protein platform on the surface of a genetically engineered microorganism such as E. coli.

According to another aspect, the present invention relates to specific preparations comprising antibodies raised against at least one of the Staphylococcus aureus and Staphylococcus epidermidis midis antigens or Staphylococcus aureus and Staphylococcus epidermidis antigen fragments as defined above. These antibodies are preferably monoclonal antibodies.

Methods for producing such antibody preparations, polyclonal or monoclonal, are well available to the man skilled in the art and properly described in the prior art. A preferred method for producing such monoclonal antibody preparation is characterized by the following steps

-   -   initiating an immune response in a non human animal by         administering a Staphylococcus antigen or a fragment thereof, as         defined above, to said animal,     -   removing the spleen or spleen cells from said animal,     -   producing hybridoma cells of said spleen or spleen cells,     -   selecting and cloning hybridoma cells specific for said antigen         and     -   producing the antibody preparation by cultivation of said cloned         hybridoma cells and optionally further purification steps.

Preferably, removing of the spleen or spleen cells is connected with killing said animal.

Monoclonal antibodies and fragments thereof can be chimerized or humanized (Graziano et al. 1995) to enable repeated administration. Alternatively human monoclonal antibodies and fragments thereof can be obtained from phage-display libraries (McGuinnes et al., 1996) or from transgenic animals (Bruggemann et al., 1996).

A preferred method for producing polyclonal antibody preparations to said Staphylococcus aureus or Staphylococcus epidermidis antigens identified with the present invention is characterized by the following steps

-   -   initiating an immune response in a non human animal by         administering a Staphylococcus antigen or a fragment thereof, as         defined above, to said animal,     -   removing an antibody containing body fluid from said animal,     -   and     -   producing the antibody preparation by subjecting said antibody         containing body fluid to further purification steps.

These monoclonal or polyclonal antibody preparations may be used for the manufacture of a medicament for treating or preventing diseases due to staphylococcal infection. Moreover, they may be used for the diagnostic and imaging purposes.

The method is further described in the following examples and in the figures, but should not be restricted thereto.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the pre-selection of sera based on anti-staphylococcal antibody titers measured by ELISA.

FIG. 2 shows the size distribution of DNA fragments in the LSA50/6 library in pMAL4.1.

FIG. 3 shows the MACS selection with biotinylated human serum. The LSA50/6 library in pMAL9.1 was screened with 10 μg biotinylated, human serum in the first (A) and with 1 μg in the second selection round (B). P.serum, patient serum; B.serum, infant serum. Number of cells selected after the 2^(nd) and 3^(rd) elution are shown for each selection round.

FIG. 4 shows the serum reactivity with specific clones isolated by bacterial surface display as analyzed by Western blot analysis with patient serum at a dilution of 1:5000.

FIG. 5 shows peptide ELISA with serum from patients and healthy individuals with an epitope identified by ribosome display.

FIG. 6 shows representative 2D Immunoblot of S. aureus surface proteins detected with human sera. 800 μg protein from S. aureus/COL grown on BHI were resolved by IEF (pI 4-7) and SDS-PAGE (9-16%), and subsequently transferred to PVDF membrane. After blocking, the membrane was incubated with sera IC35 (1:20,000). Binding of serum IgG was visualized by an anti-human IgG/HRPO conjugate and ECL development.

FIG. 7 demonstrates a representative 2D gel showing S. aureus surface proteins stained by Coomassie Blue. 1 mg protein from S. aureus/COL were resolved by IEF (pI 4-7) and SDS-PAGE (9-16%). Spots selected for sequencing after serological proteome analysis are marked.

FIGS. 8A and 8B show the structure of LPXTG (SEQ ID NO:641) cell wall proteins (SEQ ID NOS:604 through 639).

FIG. 9 shows the IgG response in uninfected (N, C) and infected (P) patients to LPXTGV (SEQ ID NO:640), a novel antigen and probable surface adhesin of S. aureus, discovered by both the inventive bacterial surface-display and proteomics approaches.

FIG. 10 shows the surface staining of S. aureus with purified anti-LPXTGV (SEQ ID NO:640) IgGs.

EXAMPLES Discovery of Novel Staphylococcus aureus Antigens Example 1 Preparation of Antibodies from Human Serum

The antibodies produced against staphylococci by the human immune system and present in human sera are indicative of the in vivo expression of the antigenic proteins and their immunogenicity. These molecules are essential for the identification of individual antigens in the approach as the present invention which is based on the interaction of the specific anti-staphylococcal antibodies and the corresponding S. aureus peptides or proteins. To gain access to relevant antibody repertoires, human sera were collected from I. patients with acute S. aureus infections, such as bacteriaemia, sepsis, infections of intravascular and percutan catheters and devices, wound infections, and superficial and deep soft tissue infection. S. aureus was shown to be the causative agent by medical microbiological tests. II. A collection of serum samples from uninfected adults was also included in the present analysis, since staphylococcal infections are common, and antibodies are present as a consequence of natural immunization from previous encounters with Staphylococci from skin and soft tissue infections (furunculus, wound infection, periodontitis etc.).

The sera were characterized for S. aureus antibodies by a series of ELISA assays. Several staphylococcal antigens have been used to prove that the titer measured was not a result of the sum of cross-reactive antibodies. For that purpose not only whole cell S. aureus (protein A deficient) extracts (grown under different conditions) or whole bacteria were used in the ELISA assays, but also individual cell wall components, such as lipoteichoic acid and peptidoglycan isolated from S. aureus. More importantly, a recombinant protein collection was established representing known staphylococcal cell surface proteins for the better characterization of the present human sera collections.

Recently it was reported that not only IgG, but also IgA serum antibodies can be recognized by the FcRIII receptors of PMNs and promote opsonization (Phillips-Quagliata et al., 2000; Shibuya et al., 2000). The primary role of IgA antibodies is neutralization, mainly at the mucosal surface. The level of serum IgA reflects the quality, quantity and specificity of the dimeric secretory IgA. For that reason the serum collection was not only analyzed for anti-staphylococcal IgG, but also for IgA levels. In the ELISA assays highly specific secondary reagents were used to detect antibodies from the high affinity types, such as IgG and IgA, and avoided IgM. Production of IgM antibodies occurs during the primary adaptive humoral response, and results in low affinity antibodies, while IgG and IgA antibodies had already under-gone affinity maturation, and are more valuable in fighting or preventing disease

Experimental Procedures

Enzyme linked immune assay (ELISA). ELISA plates were coated with 2-10 μg/ml of the different antigens in coating buffer (sodium carbonate pH 9.2). Serial dilutions of sera (100-100.000) were made in TBS-BSA. Highly specific (cross-adsorbed) HRP (Horse Radish Peroxidase)-labeled anti-human IgG or anti-human IgA secondary antibodies (Southern Biotech) were used according to the manufacturers' recommendations (˜2.000×). Antigen-antibody complexes were quantified by measuring the conversion of the substrate (ABTS) to colored product based on OD_(405 nm) readings in an automated ELISA reader (Wallace Victor 1420). The titers were compared at given dilution where the dilution response was linear (Table 1). The 100 sera were ranked based on the reactivity against multiple staphylococcal components, and the highest ones (above 90 percentile) were selected for further analysis in antigen identification. Importantly, the anti-staphylococcal antibodies from sera of clinically healthy individuals proved to be very stable, giving the same high ELISA titers against all the staphylococcal antigens measured after 3, 6 and 9 months (data not shown). In contrast, anti-S. aureus antibodies in patients decrease, then disappear after a couple of weeks following the infection (Coloque-Navarro et al, 1998). However, antibodies from patients are very important, since these are direct proof of the in vivo expression of the bacterial antigens tested in or ELISAs or identified as immunogenic during the screens according to the present invention.

This comprehensive approach followed during antibody characterization is unique, and led to unambiguous identification of anti-staphylococcal hyperimmune sera.

Purification of antibodies for genomic screening. Five sera from both the patient and the noninfected group were selected based on the overall anti-staphylococcal titers. Antibodies against E. coli proteins were removed by either incubating the heat inactivated sera with whole cell E. coli (DH5a, transformed with pHIE11, grown under the same condition as used for bacterial display) or with E. coli lysate affinity chromatography for ribosome display. Highly enriched preparations of IgG from the pooled, depleted sera were generated by protein G affinity chromatography, according to the manufacturer's instructions (UltraLink Immobilized Protein G, Pierce). IgA antibodies were purified also by affinity chromatography using biotin-labeled anti-human IgA (Southern Biotech) immobilized on Streptavidin-agarose (GIBCO BRL). The efficiency of depletion and purification was checked by SDS-PAGE, Western blotting, ELISA, and protein concentration measurements. For proteomics, the depletion the IgG and IgA preparation was not necessary, since the secondary reagent ensured the specificity.

Example 2 Generation of Highly Random, Frame-Selected, Small-Fragment, Genomic DNA Libraries of Staphylococcus aureus

Experimental Procedures

Preparation of staphylococcal genomic DNA. This method was developed as a modification of two previously published protocols (Sohail, 1998, Betley et al., 1984) and originally specifically adapted for the methicillin resistant Staphylococcus aureus strain COL to obtain genomic DNA in high quality and large scale. 500 ml BHI (Brain Heart Infusion) medium supplemented with 5 μg/ml Tetracycline was inoculated with bacteria from a frozen stab and grown with aeration and shaking for 18 h at 37° C. The culture was then harvested in two aliquots of 250 ml each, centrifuged with 1600×g for 15 min and the supernatant was removed. Bacterial pellets were carefully re-suspended in 26 ml of 0.1 mM Tris-HCl, pH 7.6 and centrifuged again with 1600×g for 15 min. Pellets were re-suspended in 20 ml of 1 mM Tris-HCl, pH 7.6, 0.1 mM EDTA and transferred into sterile 50 ml polypropylene tubes. 1 ml of 10 mg/ml heat treated RNase A and 200 U of RNase Ti were added to each tube and the solution mixed carefully. 250 μl of Lysostaphin (10 mg/ml stock, freshly prepared in ddH₂O) was then added to the tubes, mixed thoroughly and incubated at 40° C. for 10 min in a shaking water bath under continuous agitation. After the addition of 1 ml 10% SDS, 40 μl of Proteinase K (25 mg/ml stock) and 100 μl of Pronase (10 mg/ml), tubes were again inverted several times and incubated at 40° C. for 5 min in a shaking water bath. 3.75 ml of 5 M NaCl and 2.5 ml of cetyl trimethyl-ammonium bromide solution (CTAB) (10% w/v, 4% w/v NaCl) were then added and tubes were further incubated at 65° C. in a shaking water bath for 10 min. Samples were cooled to room temperature and extracted with PhOH/CHCl₃/IAA (25:24:1) and with CHCl₃/IAA (24:1). Aqueous phases were carefully collected and transferred to new sterile 50-ml tubes. To each tube 1.5 ml of Strataclean™ Resin was added, mixed gently but thoroughly and incubated for one minute at room temperature. Samples were centrifuged and the upper layers containing the DNA were collected into clean 50 ml-tubes. DNA was precipitated at room temperature by adding 0.6× volume of Isopropanol, spooled from the solution; with a sterile Pasteur pipette and transferred into tubes containing 80% ice cold ethanol. DNA was recovered by centrifuging the precipitates with 10-12,000×g, then dried on air and dissolved in ddH₂O.

Preparation of small genomic DNA fragments. Genomic DNA fragments were mechanically sheared into fragments ranging in size between 150 and 300 bp using a cup-horn sonicator (Bandelin Sonoplus UV 2200 sonicator equipped with a BB5 cup horn, 10 sec. pulses at 100% power output) or into fragments of size between 50 and 70 bp by mild DNase I treatment (Novagen). It was observed that sonication yielded a much tighter fragment size distribution when breaking the DNA into fragments of the 150-300 bp size range. However, despite extensive exposure of the DNA to ultrasonic wave-induced hydromechanical shearing force, subsequent decrease in fragment size could not be efficiently and reproducibly achieved. Therefore, fragments of 50 to 70 bp in size were obtained by mild DNase I treatment using Novagen's shotgun cleavage kit. A 1:20 dilution of DNase I provided with the kit was prepared and the digestion was performed in the presence of MnCl₂ in a 60 μl volume at 20° C. for 5 min to ensure double-stranded cleavage by the enzyme. Reactions were stopped with 2 μl of 0.5M EDTA and the fragmentation efficiency was evaluated on a 2% TAE-agarose gel. This treatment resulted in total fragmentation of genomic DNA into near 50-70 bp fragments. Fragments were then blunt-ended twice using T4 DNA Polymerase in the presence of 100 μM each of dNTPs to ensure efficient flushing of the ends. Fragments were used immediately in ligation reactions or frozen at −20° C. for subsequent use.

Description of the vectors. The vector pMAL4.1 was constructed on a pEH1 backbone (Hashemzadeh-Bonehi et al., 1998) with the Kanamycin resistance gene. In addition it harbors a b-lactamase (bla) gene cloned into the multiple cloning site. The bla gene is preceded by the leader peptide sequence of ompA to ensure efficient secretion across the cytoplasmic membrane. A Sma I restriction site serves for library insertion. The Sma I site is flanked by an upstream FseI site and a downstream NotI site which were used for recovery of the selected fragments. The three restriction sites are inserted after the ompA leader sequence in such a way that the bla gene is transcribed in the −1 reading frame resulting in a stop codon 15 bp after the NotI site. A +1 bp insertion restores the bla ORF so that b-lactamase protein is produced with a consequent gain of Ampicillin resistance.

The vector pMAL4.31 was constructed on a pASK-IBA backbone (Skerra, 1994) with the b-lactamase gene exchanged with the Kanamycin resistance gene. In addition it harbors a b-lactamase (bla) gene cloned into the multiple cloning site. The sequence encoding mature b-lactamase is preceded by the leader peptide sequence of ompA to allow efficient secretion across the cytoplasmic membrane. Furthermore a sequence encoding the first 12 amino acids (spacer sequence) of mature b-lactamase follows the ompA leader peptide sequence to avoid fusion of sequences immediately after the leader peptidase cleavage site, since e.g. clusters of positive charged amino acids in this region would decrease or abolish translocation across the cytoplasmic membrane (Kajava et al., 2000). A SmaI restriction site serves for library insertion. The SmaI site is flanked by an upstream FseI site and a downstream NotI site which were used for recovery of the selected fragment. The three restriction sites are inserted after the sequence encoding the 12 amino acid spacer sequence in such a way that the bla gene is transcribed in the −1 reading frame resulting in a stop codon 15 bp after the NotI site. A +1 bp insertion restores the bla ORF so that b-lactamase protein is produced with a consequent gain of Ampicillin resistance.

The vector pMAL9.1 was constructed by cloning the lamB gene into the multiple cloning site of pEH1. Subsequently, a sequence was inserted in lamB after amino acid 154, containing the restriction sites FseI, SmaI and NotI. The reading frame for this insertion was chosen in a way that transfer of frame-selected DNA fragments excised by digestion with FseI and NotI from plasmids pML4.1 or pMAL4.31 to plasmid pMAL9.1 will yield a continuous reading frame of lamB and the respective insert.

The vector pHIE11 was constructed by cloning the fhuA gene into the multiple cloning site of pEH1. Thereafter, a sequence was inserted in fhuA after amino acid 405, containing the restriction site FseI, XbaI and NotI. The reading frame for this insertion was chosen in a way that transfer of frame-selected DNA fragments excised by digestion with FseI and NotI from plasmids pMAL4.1 or pMAL4.31 to plasmid pHIE11 will yield a continuous reading frame of fhuA and the respective insert.

Cloning and evaluation of the library for frame selection. Genomic S. aureus DNA fragments were ligated into the SmaI site of either the vector pMAL4.1 or pMAL4.31. Recombinant DNA was electroporated into DH10B electrocompetent E. coli cells (GIBCO BRL) and transformants plated on LB-agar supplemented with Kanamycin (50 μg/ml) and Ampicillin (50 μg/ml). Plates were incubated over night at 37° C. and colonies collected for large scale DNA extraction. A representative plate was stored and saved for collecting colonies for colony PCR analysis and large-scale sequencing. A simple colony PCR assay was used to initially determine the rough fragment size distribution as well as insertion efficiency. From sequencing data the precise fragment size was evaluated, junction intactness at the insertion site as well as the frame selection accuracy (3n+1 rule).

Cloning and evaluation of the library for bacterial surface display. Genomic DNA fragments were excised from the pMAL4.1 or pMAL4.31 vector, containing the S. aureus library with the restriction enzymes FseI and NotI. The entire population of fragments was then transferred into plasmids pMAL9.1 (LamB) or pHIE11 (FhuA) which have been digested with FseI and NotI. Using these two restriction enzymes, which recognise an 8 bp GC rich sequence, the reading frame that was selected in the pMAL4.1 or pMAL4.31 vector is maintained in each of the platform vectors. The plasmid library was then transformed into E. coli DH5a cells by electroporation. Cells were plated onto large LB-agar plates supplemented with 50 μg/ml Kanamycin and grown over night at 37° C. at a density yielding clearly visible single colonies. Cells were then scraped off the surface of these plates, washed with fresh LB medium and stored in aliquots for library screening at −80° C.

Results

Libraries for frame selection. Two libraries (LSA50/6 and LSA250/1) were generated in the pMAL4.1 vector with sizes of approximately 50 and 250 bp, respectively. For both libraries a total number of clones after frame selection of 1-2×10⁶ was received using approximately 1 μg of pMAL4.1 plasmid DNA and 50 ng of fragmented genomic S. aureus DNA. To assess the randomness of the LSA50/6 library, 672 randomly chosen clones were sequenced. The bioinformatic analysis showed that of these clones none was present more than once. Furthermore, it was shown that 90% of the clones fell in the size range of 19 to 70 bp with an average size of 25 bp (FIG. 2). All 672 sequences followed the 3n+1 rule, showing that all clones were properly frame selected.

Bacterial surface display libraries. The display of peptides on the surface of E. coli required the transfer of the inserts from the LSA50/6 library from the frame selection vector pMAL4.1 to the display plasmids pMAL9.1 (LamB) or pHIE11 (FhuA). Genomic DNA fragments were excised by FseI and NotI restriction and ligation of 5 ng inserts with 0.1 μg plasmid DNA resulted in 2-5×10⁶ clones. The clones were scraped off the LB plates and frozen without further amplification.

Example 3 Identification of Highly Immunogenic Peptide Sequences from S. aureus Using Bacterial Surface Displayed Genomic Libraries and Human Serum

Experimental Procedures

MACS screening. Approximately 2.5×10⁸ cells from a given library were grown in 5 ml LB-medium supplemented with 50 μg/ml Kanamycin for 2 h at 37° C. Expression was induced by the addition of 1 mm IPTG for 30 min. Cells were washed twice with fresh LB medium and approximately 2×10⁷ cells re-suspended in 100 μl LB medium and transferred to an Eppendorf tube.

10 μg of biotinylated, human serum was added to the cells and the suspension incubated over night at 4° C. with gentle shaking. 900 μl of LB medium was added, the suspension mixed and subsequently centrifuged for 10 min at 6000 rpm at 4° C. Cells were washed once with 1 ml LB and then re-suspended in 100 μl LB medium. 10 μl of MACS microbeads coupled to streptavidin (Miltenyi Biotech, Germany) were added and the incubation continued for 20 min at 4° C. Thereafter 900 μl of LB medium was added and the MACS microbead cell suspension was loaded onto the equilibrated MS column (Miltenyi Biotech, Germany) which was fixed to the magnet. (The MS columns were equilibrated by washing once with 1 ml 70% EtOH and twice with 2 ml LB medium.)

The column was then washed three times with 3 ml LB medium. The elution was performed by removing the magnet and washing with 2 ml LB medium. After washing the column with 3 ml LB medium, the 2 ml eluate was loaded a second time on the same column and the washing and elution process repeated. The loading, washing and elution process was performed a third time, resulting in a final eluate of 2 ml.

A second round of screening was performed as follows. The cells from the final eluate were collected by centrifugation and resuspended in 1 ml LB medium supplemented with 50 μg/ml Kanamycin. The culture was incubated at 37° C. for 90 min and then induced with 1 mM IPTG for 30 min. Cells were subsequently collected, washed once with 1 ml LB medium and suspended in 10 μl LB medium. Since the volume was reduced, 1 μg of human, biotinylated serum was added and the suspension incubated over night at 4° C. with gentle shaking. All further steps were exactly the same as in the first selection round. Cells selected after two rounds of selection were plated onto LB-agar plates supplemented with 50 μg/ml Kanamycin and grown over night at 37° C.

Evaluation of selected clones by sequencing and Western blot analysis. Selected clones were grown over night at 37° C. in 3 ml LB medium supplemented with 50 μg/ml Kanamycin to prepare plasmid DNA using standard procedures. Sequencing was performed at MWG (Germany) or in a collaboration with TIGR (U.S.A.).

For Western blot analysis approximately 10 to 20 μg of total cellular protein was separated by 10% SDS-PAGE and blotted onto HybondC membrane (Amersham Pharmacia Biotech, England). The LamB or FhuA fusion proteins were detected using human serum as the primary antibody at a dilution of 1:5000 and anti human IgG antibodies coupled to HRP at a dilution of 1:5000 as secondary antibodies. Detection was performed using the ECL detection kit (Amersham Pharmacia Biotech, England). Alternatively, rabbit anti FhuA or mouse anti LamB antibodies were used as primary antibodies in combination with the respective secondary antibodies coupled to HRP for the detection of the fusion proteins.

Results

Screening of bacterial surface display libraries by magnetic activated cell sorting (MACS) using biotinylated human serum. The libraries LSA50/6 in pMAL9.1 and LSA250/1 in pHIE11 were screened with a pool of biotinylated, human patient sera (see Example 1) Preparation of antibodies from human serum). The selection procedure was performed as described under Experimental procedures. As a control, pooled human sera from infants that have most likely not been infected with S. aureus was used. Under the described conditions between 10 and 50 fold more cells with the patient compared to the infant serum were routinely selected (FIG. 3). To evaluate the performance of the screen, approximately 100 selected clones were picked randomly and subjected to Western blot analysis with the same pooled patient serum. This analysis revealed that 30 to 50% of the selected clones showed reactivity with antibodies present in patient serum whereas the control strain expressing LamB or FhuA without a S. aureus specific insert did not react with the same serum. Colony PCR analysis showed that all selected clones contained an insert in the expected size range.

Subsequent sequencing of a larger number of randomly picked clones (500 to 800 per screen) led to the identification of the gene and the corresponding peptide or protein sequence that was specifically recognized by the human patient serum used for screening. The frequency with which a specific clone is selected reflects at least in part the abundance and/or affinity of the specific antibodies in the serum used for selection and recognizing the epitope presented by this clone. In that regard it is striking that some clones (ORF2264, ORF1951, ORF0222, lipase and IsaA) were picked up to 90 times, indicating their highly immunogenic property. All clones that are presented in Table 2 have been verified by Western blot analysis using whole cellular extracts from single clones to show the indicated reactivity with the pool of human serum used in the screen.

It is further worth noticing that most of the genes identified by the bacterial surface display screen encode proteins that are either attached to the surface of S. aureus and/or are secreted. This is in accordance with the expected role of surface attached or secreted proteins in virulence of S. aureus.

Assessment of reactivity of highly immunogenic peptide sequences with different human sera. 10 to 30 different human patient sera were subsequently used to evaluate the presence of antibodies against the selected immunogenic peptide sequences that have been discovered in the screen according to the present invention. To eliminate possible cross-reactivity with proteins expressed by E. coli, all sera were pre-adsorbed with a total cellular lysate of E. coli DHa cells expressing FhuA protein.

This analysis is summarized in Table 2 and as an example shown in FIG. 4 and is indicative of the validity of the present screen. It further shows that already short selected epitopes can give rise to the production of antibodies in a large number of patients (ORF1618, ORF1632, IsaA, Empbp, Protein A). Those peptide sequences that are not recognized by a larger set of patient sera may still be part of an highly immunogenic protein, but the recombinant protein itself may be tested for that purpose for each single case.

Example 4 Identification of Highly Immunogenic Peptide Sequences from Genomic Fragments from S. aureus Using Ribosome Display and Human Serum

Experimental Procedures

Ribosome display screening: 2.4 ng of the genomic library from S. aureus LSA 250/1 in pMAL4.1 (described above) was PCR amplified with oligos ICC277 and ICC202 in order to be used for ribosome display. Oligos ICC277 (CGAATAATACGACTCACTATAGGGAGACCACAACGGTTTCCCACTAG TAATAATTGTTTAACTTTAAGAAGGAGATATATCCATGCAGACCTTG GCCGGCCTCCC—SEQ ID NO:600) and ICC202 (GGCCCACCCGTGAAGGTGAGCCGGCGTAAGATGCTTTTCTGTGACTG G—SEQ ID NO:601) hybridize 5′ and 3′ of the Fse I-Not I insertion site of plasmid pMAL4.1, respectively. ICC277 introduces a T7 phage RNA polymerase promoter, a palindromic sequence resulting in a stem-loop structure on the RNA level, a ribosome binding site (RBS) and the translation start of gene 10 of the T7 phage including the ATG start codon.

Oligo ICC202 hybridizes at nucleotide position 668 of the β-lactamase open reading frame and also introduces a stem-loop structure at the 3′ end of the resulting RNA. PCR was performed with the High fidelity PCR kit (Roche Diagnostic) for 25 cycles at 50° C. hybridization temperature and otherwise standard conditions.

The resulting PCR library was used in 5 consecutive rounds of selection and amplification by ribosome display similar as described previously (Hanes et al., 1997) but with modifications as described below.

One round of ribosome display contained the following steps: In vitro transcription of 2 μg PCR product with the RiboMax kit (Promega) resulted in ca. 50 μg A. In vitro translation was performed for 9 minutes at 37° C. in 22 μl volume with 4.4 μl Premix Z (250 mM TRIS-acetate pH 7.5, 1.75 mM of each amino acid, 10 mm ATP, 2.5 mM GTP, 5 mM cAMP, 150 mM acetylphosphate, 2.5 mg/ml E. coli tRNA, 0.1 mg/ml folinic acid, 7.5% PEG 8000, 200 mM potassium glutamate, 13.8 mM Mg(Ac)2, 8 μl S30 extract (x mg/ml) and about 2 μg in vitro transcribed RNA from the pool. S30 extract was prepared as described (Chen et al, 1983). Next, the sample was transferred to an ice-cold tube containing 35.2 μl 10% milk-WBT (TRIS-acetate pH 7.5, 150 mM NaCl, 50 mM Mg(Ac)2, 0.1% TWEEN-20 (polyethylene glycol sorbitan monolaurate), 10% milk powder) and 52.8 μl WBTH (as before plus 2.5 mg/ml heparin). Subsequently, immuno precipitation was performed by addition of 10 μg purified IgGs, incubation for 90 minutes on ice, followed by addition of 30 μl MAGmol Protein G beads (Miltenyi Biotec, 90 minutes on ice). The sample was applied to a pre-equilibrated μ column (Miltenyi Biotec) and washed 5 times with ice-cold WBT buffer. Next 20 μl EB20 elution buffer (50 mM TRIS-acetate, 150 mM NaCl, 20 mM EDTA, 50 μg/ml S. cerevisiae RNA) was applied to the column, incubated for 5 minutes at 4° C. Elution was completed by adding 2×50 μl EB20. The mRNA from the elution sample was purified with the High pure RNA isolation kit (Roche Diagnostics). Subsequent reverse transcription was performed with Superscript II reverse transcriptase kit (Roche Diagnostics) according to the instruction of the manufacturer with 60 pmol oligo ICC202 for 1 hour at 50° C. in 50 μl volume. 5 μl of this mix was used for the following PCR reaction with primers ICC202 and ICC277 as described above.

Three rounds of ribosome display were performed and the resulting selected PCR pool subsequently cloned into plasmid pHIE11 (described above) by cleavage with restriction endonucleases NotI and FseI.

Evaluation of selected clones by sequencing and peptide-ELISA analysis: Selected clones were grown over night at 37° C. in 3 ml LB medium supplemented with 50 μg/ml Kanamycin to prepare plasmid DNA using standard procedures. Sequencing was performed at MWG (Germany) or at the Institute of Genomic Research (TIGR; Rockville, Md., U.S.A.). Peptides corresponding to the inserts were synthesized and coated in 10 mM NaHCO₃ pH 9.3 at a concentration of 10 μg/ml (50 μl) onto 96-well microtiter plates (Nunc). After blocking with 1% BSA in PBS at 37° C., 1:200 and 1:1000 dilutions of the indicated sera were diluted in 1% BSA/PBS and applied to the wells. After washing with PBS/0.1% TWEEN-20 (polyethylene glycol sorbitan monolaurate), biotin-labeled anti-human IgG secondary antibodies (SBA) were added and these were detected by subsequent adding horseradish-peroxidase-coupled streptavidin according to standard procedures.

Results

The 250-bp genomic library (LSA250/1) as described above was used for screening. Purified IgGs from uninfected adults but with high titer against S. aureus as described above were used for selection of antigenic peptides.

Three rounds of ribosome display selection and amplification were performed according to Experimental procedures; finished by cloning and sequencing the resulting PCR pool.

Sequence analyses of a large number of randomly picked clones (700) led to the identification of the gene and the corresponding peptide or protein sequence that was specifically recognized by the high titer serum used for screening. The frequency with which a specific clone was selected reflects at least in part the abundance and/or affinity of the specific antibodies in the serum used for selection and recognizing the epitope presented by this clone. Remarkably, some clones (ORFs) were picked up to 50 times, indicating their highly immunogenic property. Table 2 shows the ORF name, the Seq. ID No. and the number of times it was identified by the inventive screen.

For a number of immuno-selected ORFs peptides corresponding to the identified immunogenic region were synthesized and tested in peptide-ELISA for their reactivity towards the sera pool they were identified with and also a number of additional sera from patients who suffered from an infection by S. aureus. The two examples in the graphs in FIG. 5 show the values of peptides from aureolysin and Pls. They are not only hyperimmune reactive against the high titer sera pool but also towards a number of individual patient's sera. All synthesized peptides corresponding to selected immunogenic regions showed reactivity towards the high titer sera pool and Table 2 summarizes the number of times the peptides were reactive towards individual patients sera, similar as described above.

In addition, it is striking that for those ORFs that were also identified by bacterial surface display described above), very often the actual immunogenic region within the ORF was identical or overlapping with the one identified by ribosome display. This comparison can be seen in Table 2.

Example 5 Identification of Highly Immunogenic Antigens from S. aureus Using Serological Proteome Analysis

Experimental Procedures

Surface protein preparations from S. aureus containing highly immunogenic antigens. S. aureus strains COL (Shafer and Iandolo, 1979) and, agr- (Recsei et al., 1986) were stored as glycerol stocks at −80° C. or on BHI (DIFCO) plates at 4° C. Single clones were used for inoculation of overnight cultures in either BHI (“standard conditions”) or RPMI 1640 (GibcoBRL), last one depleted from iron (“stress conditions” a) by treating o/n with iminodiacetic acid (Sigma). Fresh medium was inoculated 1:100 the next day and bacteria were grown to O.D.₆₀₀ between 0.3 and 0.7. Bacteria were harvested by centrifugation and washed with ice-cold PBS. Surface proteins were prepared by lysostaphin treatment under isotonic conditions (Lim et al. 1998). Briefly; ˜3×10⁹ bacteria (according to O.D.₆₀₀=1 are about 5×10⁷ bacteria) were resuspended in 1 ml digestion buffer containing 35% raffinose (Aldrich Chemical Company), protease inhibitors (Roche) and 5 units lysostaphin (Sigma). After incubation at 37° C. for 30 min, protoplasts were carefully sedimented by low-speed centrifugation. This treatment releases surface proteins covalently linked to the pentaglycine bridge of the peptidoglycan cell wall to the supernatant (in Crossley, 1997). Cell surface proteins were either precipitated with methanol/chlorophorm (Wessel, 1984) or concentrated in centrifugal filter-tubes (Millipore). Protein samples were frozen and stored at −80° C. or dissolved in sample buffer and used for isoelectric focusing (IEF) immediately (Pasquali et al. 1997).

Serological proteome analysis of surface protein preparations from S. aureus. Samples were obtained from a) S. aureus/agr grown under “stress conditions”, b) S. aureus/COL grown under “standard conditions” and c) S. aureus/COL “stress conditions”. Loading onto 17 cm-strips containing immobilized pH gradients (pH 4-7, BioRad) was done using the “in-gel-reswelling procedure” (Pasquali et al., 1997). The gels for blotting were loaded with 100-800 μg protein, the preparative gels with 400-1,000 μg protein. Isoelectric focusing and SDS-PAGE (9-16% gradient gels) were performed as described (Pasquali et al., 1997). For Western blotting, proteins were transferred onto PVDF-membranes (BioRad) by semi-dry blotting. Transfer-efficiency was checked by amido-black staining. After blocking (PBS/0.1% TWEEN-20 (polyethylene glycol sorbitan monolaurate)/10% dry milk, 4° C. for 16 h), blots were incubated for two hours with serum (1:2,500-1:100,000 in blocking solution, see Table 3). After washing, specific binding of serum IgG was visualized with a goat-anti-human-IgG/peroxidase conjugate (1:25,000, Southern Biotech) as secondary antibody and development with a chemiluminescence substrate (ECLE, Amersham). A representative result is shown in FIG. 6. Membranes were stripped by treatment with 2% β-ME/Laemmli buffer for 30 min at 50-65° C., immediately re-probed with a different serum, and developed as described above. This procedure was repeated up to five times. Signals showing up with patient and/or healthy donor control sera but not with the infant pool, were matched to the Coomassie (BioRad) stained preparative gels (example shown in FIG. 7). The results of these serological proteome analyses of surface protein preparations from S. aureus are summarized in Table 3.

Sequencing of protein spots by peptide-fingerprint MALDI-TOF-MS and tandem MS/MS. Gel pieces were washed alternately three times with 10 μl digestion buffer (10 mM NH₄HCO₃/CAN, 1:1). Afterwards the gel pieces were shrunken with 10 μl ACN and reswollen with 2 μl protease solution (0.05 μg/μl trypsin, Promega, Madison, USA). Digestion was performed for 10-12 h at 37° C. For MALDI-TOF-MS peptides were extracted from the gel pieces with 10 μl digestion buffer. The supernatant was concentrated with ZipTip™ (Millipore, Bedford, USA), the peptides were eluted onto the MALDI target with 0.5 μl extraction buffer (0.1% TFA/CAN, 1:1) and 0.5 μl matrix solution (HCCA in ACN/0.1% TFA, 1:1) was added. MALDI-TOF-MS was done using a REFLEX III (Bruker Daltonik, Bremen, Germany) equipped with a SCOUT384 ion source. The acceleration voltage was set to 25 kV, and the reflection voltage to 28.7 kV. The mass range was set from 700 Da to 4000 Da. Data acquisition was done on a SUN Ultra using XACQ software, version 4.0. Post-analysis data processing was done using XMASS software, version 4.02 (Bruker Daltonik, Bremen, Germany). The results are summarized in tables 3 and 4.

Example 6 Characterisation of Highly Immunogenic Proteins from S. aureus

The antigens identified by the different screening methods with the IgG and IgA preparations form pre-selected sera are further characterized, by the following ways:

1. The proteins are purified, most preferably as recombinant proteins expressed in E. coli or in a Gram+ expression system or in an in vitro translation system, and evaluated for antigenicity by a series of human sera. The proteins are modified based on bioinformatic analysis: N-terminal sequences representing the signal peptide are removed, C-terminal regions downstream of the cell wall anchor are also removed, and extra amino acids as tags are introduced for the ease of purification (such as Strep-tagII, His-tag, etc.) A large number of sera is then used in ELISA assays to assess the fraction of human sera containing specific antibodies against the given protein (see FIG. 9 as an example). One of the selected antigens is a 895 aa long protein, what was called LPXTGV (SEQ ID NO:640) (see Tables 2 and 4), since it contains the Gram+ cell wall anchor sequence LPXTG (SEQ ID NO:641). This signature has been shown to serve as cleavage site for sortase, a trans-peptidase which covalently links LPXTG (SEQ ID NO:641) motif containing proteins to the peptidoglycan cell wall. LPXTGV (SEQ ID NO:640) is also equipped with a typical signal peptide (FIG. 8). ELISA data using this protein as a Strep-tagged recombinant protein demonstrate that this protein is highly immunogenic (high titers relative to other recombinant proteins) in a high percentage of sera (FIG. 9). Importantly, patients with acute S. aureus infection produce significantly more of these anti-LPXTGV (SEQ ID NO:640) antibodies, than healthy normals, suggesting that the protein is expressed during in vivo infection. The overall ELISA titers of the individual antigenic proteins are compared, and the ones inducing the highest antibody levels (highly immunogenic) in most individuals (protein is expressed by most strains in vivo) are favored. Since the antigen specificity and quality (class, subtype, functional, nonfunctional) of the antibodies against S. aureus produced in individual patients can vary depending on the site of infection, accompanying chronic diseases (e.g. diabetes) and chronic conditions (e.g. intravascular device), and the individuals' immune response, special attention was paid to the differences detected among the different patient groups, since medical records belonging to each sera were available. In addition, each patient serum is accompanied by the pathogenic strain isolated from the patient at the time of serum sampling.

Specific antibodies are purified for functional characterization. The purity and the integrity of the recombinant proteins are checked (e.g. detecting the N-terminal Strep-tag in Western blot analysis in comparison to silver staining in SDS-PAGE). The antigens are immobilized through the tags to create an affinity matrix, and used for the purification of specific antibodies from highly reactive sera. Using as an example strep-tagged LPXTGV (SEQ ID NO:640) as the capture antigen, 20 μg of antibody from 125 mg of IgG were purified. Based on the ELISA data a pure preparation was received, not having e.g. anti-LTA and anti-peptidoglycan (both dominant with unfractionated IgG) activity. The antibodies are then used to test cell surface localization by FACS and fluorescent microscopy (FIG. 10).

3. Gene Occurrence in Clinical Isolates

An ideal vaccine antigen would be an antigen that is present in all, or the vast majority of, strains of the target organism to which the vaccine is directed. In order to establish whether the genes encoding the identified Staphylococcus aureus antigens occur ubiquitously in S. aureus strains, PCR was performed on a series of independent S. aureus isolates with primers specific for the gene of interest. S. aureus isolates were obtained from patients with various S. aureus infections. In addition several nasal isolates from healthy carriers and several lab strains were also collected and analyzed. The strains were typed according to restriction fragment length polymorphism (RFLP) of the spa and coa genes (Goh et al. 1992, Frénay et al., 1994, vanden Bergh et al. 1999). From these results 30 different strains were identified—24 patient isolates, 3 nasal isolates and 3 lab strains. To establish the gene distribution of selected antigens, the genomic DNA of these 30 strains was subjected to PCR with gene specific primers that flank the selected epitope (ORF1361: Seq. ID No. 187 and 188; ORF2268: Seq. ID No. 193 and 194; ORF1951: Seq. ID No. 195 and 196; ORF1632: Seq. ID No. 181 and 182; ORF0766: Seq. ID No. 183 and 184; ORF0576: Seq. ID No. 185 and 186; ORF0222: Seq. ID No. 189 and 190; ORF0360: Seq. ID No. 191 and 192). The PCR products were analyzed by gel electrophoresis to identify a product of the correct predicted size. ORFs 1361, 2268, 1951, 1632, 0766 and 0222 are present in 100% of strains tested and ORF0576 in 97%. However ORF0360 occurred in only 71% of the strains. Thus ORFs 1361, 2268, 1951, 1632, 0766, 0576 and 0222 each have the required ubiquitous presence among S. aureus isolates.

These antigens (or antigenic fragments thereof, especially the fragments identified) are especially preferred for use in a vaccination project against S. aureus.

4. Identification of Highly Promiscuous HLA-Class II Helper Epitopes within the ORFs of Selected Antigens

The ORFs corresponding to the antigens identified on the basis of recognition by antibodies in human sera, most likely also contain linear T-cell epitopes. Especially the surprising finding in the course of the invention that even healthy uninfected, non-colonized individuals show extremely high antibody titers (>100,000 for some antigens, see Example 5) which are stable for >1 year (see Example 1), suggests the existence of T-cell dependent memory most probably mediated by CD4+ helper-T-cells. The molecular definition of the corresponding HLA class II helper-epitopes is useful for the design of synthetic anti-staphylococcal vaccines, which can induce immunological memory. In this scenario the helper-epitopes derived from the staphylococcal antigens provide “cognate help” to the B-cell response against these antigens or fragments thereof. Moreover it is possible to use these helper-epitopes to induce memory to T-independent antigens like for instance carbohydrates (conjugate vaccines). On the other hand, intracellular occurring staphylococci can be eliminated by CD8+ cytotoxic T-cells, which recognize HLA class I restricted epitopes.

T-cell epitopes can be predicted by various public domain algorithms: http://bimas.dcrt.nih.aov/molbio/hla bind/ (Parker et al. 1994), http://134.2.96.221/scripts/MHCServer.dll/home.htm (Rammensee at al. 1999), http://mypage.ihost.com/usinet.hamme76/ (Sturniolo et al. 1999). The latter prediction algorithm offers the possibility to identify promiscuous helper-epitopes, i.e. peptides that bind to several HLA class II molecules. In order to identify highly promiscuous helper-epitopes within staphylococcal antigens the ORFs corresponding to Seq ID 64 (IsaA), Seq ID 114 (POV2), Seq ID 89 (QRF0222), Seq ID 70 (LPXTGIV), Seq ID 56 (LPXTGV), Seq ID 142 (LPXTGVI), Seq ID 81 (ORF3200), Seq ID 74 (ORF1951), Seq ID 94 (Empbp), Seq ID 83 (autolysin) and Seq ID 58 (ORF2498) were analyzed using the TEPITOPE package http://mypage.ihost.com/usinet.hamme76/ (Sturniolo et al. 1999). The analysis was done for 25 prevalent DR-alleles and peptides were selected if they were predicted to be a) strong binders (1% threshold) for at least 10/25 alleles or b) intermediate (3% threshold) binders for at least 17/25 alleles.

The following peptides containing one or several promiscuous helper-epitopes were selected (and are claimed):

Seq ID 56: pos. 6-40, 583-598, 620-646, 871-896 Seq ID 58: no peptide fulfills selection criteria Seq ID 64: no peptide fulfills selection criteria Seq ID 70: pos. 24-53 Seq ID 74: pos. 240-260 Seq ID 81: pos. 1660-1682, 1746-1790 Seq ID 83: pos. 1-29, 680-709, 878-902 Seq ID 89: pos. 96-136 Seq ID 94: pos. 1-29, 226-269, 275-326 Seq ID 114: pos. 23-47, 107-156 Seq ID 142: pos. 24-53

The corresponding peptides or fragments thereof (for instance overlapping 15-mers) can be synthesized and tested for their ability to bind to various HLA molecules in vitro. Their immunogenicity can be tested by assessing the peptide (antigen)-driven proliferation (BrdU or 3H-thymidine incorporation) or the secretion of cytokines (ELIspot, intracellular cytokine staining) of T-cells in vitro (Mayer et al. 1996, Schmittel et al. 2000, Sester et al. 2000). In this regard it will be interesting to determine quantitative and qualitative differences in the T-cell response to the staphylococcal antigens or the selected promiscuous peptides or fragments thereof in populations of patients with different staphylococcal infections, or colonization versus healthy individuals neither recently infected nor colonized. Moreover, a correlation between the antibody titers and the quantity and quality of the T-cell response observed in these populations is expected. Alternatively, immunogenicity of the predicted peptides can be tested in HLA-transgenic mice (Sonderstrup et al. 1999).

Similar approaches can be taken for the identification of HLA class I restricted epitopes within staphylococcal antigens.

Synthetic Peptides Representing One or More Promiscuous T Helper Epitopes from S. aureus

Partially overlapping peptides spanning the indicated regions of Seq ID 56 (LPXTGV), Seq ID 70 (LPXTGIV), Seq ID 74 (ORF1hom1), Seq ID 81 (EM_BP), Seq ID 83 (Autolysin), Seq ID 89 (ORF1hom2), Seq ID 94 (EMPBP), Seq ID 114 (POV2) and Seq ID 142 (LPXTGVI) were synthesized. Sequences of the individual peptides are given in Table 5. All peptides were synthesized using Fmoc chemistry, HPLC purified and analyzed by mass spectrometry. Lyophilized peptides were dissolved in DMSO and stored at −20° C. at a concentration of 5-10 mM.

Binding of Synthetic Peptides Representing Promiscuous T Helper Epitopes to HLA Molecules In Vitro

Binding of peptides to HLA molecules on the surface of antigen-presenting cells is a prerequisite for activation of T cells. Binding was assessed in vitro by two independent biochemical assays using recombinant soluble versions of HLA class II molecules. One assay measures the concentration dependent competitive replacement of a labeled reference peptide by the test peptides. The second assay is based on the formation of SDS-stable complexes upon binding of high- and intermediate affinity ligands. A summary of the results obtained by the two assays is given in Table 5.

Soluble HLA molecules (DRA1*0101/DRB1*0101 and DRA1*0101/DRB1*0401) were expressed in SC-2 cells and purified as described in Aichinger et al., 1997. For the competition assay (Hammer et al. 1995) HLA molecules were applied between 50 and 200 ng/well. For DRB1*0101 biotinilated indicator peptide HA (PKYVKQNTLKLAT—SEQ ID NO:602, Valli et al. 1993) was used at 0.008 μM. For DRB1*0401 biotinilated indicator peptide UD4 (YPKFVKQNTLKAA—SEQ ID NO:603, Valli et al. 1993) was used between 0.03 and 0.06 μM. Molecules, indicator and test peptides were incubated overnight at 37° C., pH 7. HLA:peptide complexes obtained after incubation with serial dilutions of test and reference peptides (the known high-affinity binders HA and UD4 were used as positive control) were captured in ELISA plates coated with antibody L243, which is known to recognize a conformational epitope formed only by correctly associated heterodimers. Incorporated biotin was measured by standard colorimetric detection using a streptavidin-alkaline phosphatase conjugate (Dako) with NBT/BCIP tablets (Sigma) as substrate and automated OD reading on a Victor reader (Wallac).

T Cell Response Against Promiscuous T Helper Epitopes Assessed by IFNg ELIspot Assay

Upon antigenic stimulation T cells start to proliferate and to secrete cytokines such as interferon gamma (IFNg). Human T cells specifically recognizing epitopes within S. aureus antigens were detected by IFNg-ELIspot (Schmittel et al. 2000). PBMCs from healthy individuals with a strong anti-S. aureus IgG response were isolated from 50-100 ml of venous blood by ficoll density gradient centrifugation and used after freezing and thawing. Cells were seeded at 200,000/well in 96-well plates. Peptides were added as mixtures corresponding to individual antigens, in both cases at 10 μg/ml each. Concanavalin A (Amersham) and PPD (tuberculin purified protein derivate, Statens Serum Institute) served as assay positive controls, assay medium without any peptide as negative control. After overnight incubation in Multi Screen 96-well filtration plates (Millipore) coated with the anti-human IFNg monoclonal antibody B140 (Bender Med Systems) the ELIspot was developed using the biotinylated anti-human IFNg monoclonal antibody B308-BT2 (Bender Med Systems), Streptavidin-alkaline phosphatase (DAKO) and BCIP/NBT alkaline phosphatase substrate (SIGMA). Spots were counted using an automatic plate reader (Bioreader 2000, BIO-SYS). Spots counted in wells with cells stimulated with assay medium only (negative control, generally below 10 spots/100,000 cells) were regarded as background and subtracted from spot numbers counted in wells with peptides.

TABLE 5 Promiscuous T helper epitopes contained in S. aureus antigens Amino acid sequences within S. aureus antigens containing IFNg highly promiscuous T helper epitopes binding ¹⁾ ELIspot ²⁾ Seq ID 56 (LPXTGV): pos. 6-40 p6-28  >PKLRSFYSIRKSTLGVASVIVST// + 44; 80; 8; p24-40                    >VIVSTLFLISQHQAQA// − 95; 112 Seq ID 56 (LPXTGV): pos. 620-646 p620-646  >FPYIPDKAVYNAIVKVVVANIGYEGQ// + Seq ID 56 (LPXTGV): pos. 871-896 p871-896  >QSWWGLYALLGMLALFIPKFRKESK// − Seq ID 70 (LPXTGIV): pos. 24-53 p24-53  >YSIRKFTVGTASILIGSLMYLGTQQEAEA// nd 34; 14; 0; 57; 16 Seq ID 74 (ORF1hom1): pos. 240-260 p240-260  >MNYGYGPGVVTSRTISASQA// + 47; 50; 0; 85; 92 Seq ID 81 (EM_BP): pos. 1660-1682 p1660-1682  >NEIVLETIRDINNAHTLQQVEA// nd 2; 14; 5; 77; 26 Seq ID 81 (EM_BP): pos. 1746-1790 p1746-1773  >LHMRHFSNNFGNVIKNAIGVVGISGLLA// nd p1753-1779         >NNFGNVIKNAIGVVGISGLLASFWFFI// nd p1777-1789                                 >FFIAKRRRKEDEE/ nd Seq ID 83 (Autolysin) pos. 1-29 p1-29:  >MAKKFNYKLPSMVALTLVGSAVTAHQVQA// nd 6; 35; 7; 60; 49 Seq ID 83 (Autolysin) pos. 878-902 p878-902:  >NGLSMVPWGTKNQVILTGNNIAQG/ nd Seq ID 89 (ORF1hom2): pos. 96-136 p96-121  >GESLNIIASRYGVSVDQLMAANNLRG// − 0; 35; 0; p117-136                       >NNLRGYLIMPNQTLQIPNG// − 29; 104 Seq ID 94 (EMPBP): pos. 1-29 p4-29:  >KLLVLTMSTLFATQIMNSNHAKASV// + Seq ID 94 (EMPBP): pos. 226-269 p226-251  >IKINHFCVVPQINSFKVIPPYGHNS// − 26; 28; 1 p254-270  >MHVPSFQNNTTATHQN// + 6; 43; 97 Seq ID 94 (EMPBP): pos. 275-326 p275-299  >YDYKYFYSYKVVKGVKKYFSFSQS// + p284-305          >YKVVKGVKKYFSFSQSNGYKIG// + p306-326  >PSLNIKNVNYQYAVPSYSPT// + Seq ID 114 (POV2): pos. 23-47 p23-47 >AGGIFYNQTNQQLLVLCDGMGGHK// − 49; 20; 4; 77; 25 Seq ID 114 (POV2): pos. 107-156 p107-124 >ALVFEKSVVIANVGDSRA/ − p126-146 >RAYVINSRQIEQITSDHSFVN// nd p142-158                  >SFVNHLVLTGQITPEE// nd Seq ID 142 (LPXTGVI): pos. 1-42 p6-30 >KEFKSFYSIRKSSLGVASVAISTL// + + 0; 41; 20; p18-42            >SSLGVASVAISTLLLLMSNGEAQA// nd 88; 109 Seq ID 142 (LPXTGVI): pos. 209-244 p209-233 >IKLVSYDTVKDYAYIRFSVSNGTKA// + p218-244          >KDYAYIRFSVSNGTKAVKIVSSTHFNN// + Seq ID 142 (LPXTGVI): pos. 395-428 p395-418 >FMVEGQRVRTISTYAINNTRCTIF// − p416-428                      >TIFRYVEGKSLYE// − Seq ID 142 (LPXTGVI): pos. 623-647 p623-647 >MTLPLMALLALSSIVAFVLPRKRKN // − ¹⁾ binding to soluble DRA1*0101/DRB1*0401 molecules was determined using a competition assay (+, + +: binding, −: no competition up to 200 μM test peptide; nd: not done) ²⁾ results from 5 healthy individuals with strong anti-S. aureus IgG response. Data are represented as spots/200.000 cells (background values are subtracted 5. Antigens May be Injected into Mice—and the Antibodies Against these Proteins can be Measured. 6. Protective Capacity of the Antibodies Induced by the Antigens Through Vaccination can be Assessed in Animal Models.

Both 5. and 6. are methods well available to the skilled man in the art.

Example 7 Applications

A) An effective vaccine offers great potential for patients facing elective surgery in general, and those receiving endovascular devices, in particular. Patients suffering from chronic diseases with decreased immune responses or undergoing continuous ambulatory peritoneal dialysis are likely to benefit from a vaccine with S. aureus by immunogenic serum-reactive antigens according to the present invention. Identification of the relevant antigens will help to generate effective passive immunization (humanized monoclonal antibody therapy), which can replace human immunoglobulin administration with all its dangerous side-effects. Therefore an effective vaccine offers great potential for patients facing elective surgery in general, and those receiving endovascular devices, in particular.

S. aureus can cause many different diseases.

-   1. Sepsis, bacteriaemia -   2. Haemodialysed patients—bacteriemia, sepsis -   3. Peritoneal dialyses patients—peritonitis -   4. Patients with endovascular devices (heart surgery,     etc)—endocarditis, bacteriemia, sepsis -   5. Orthopedic patients with prosthetic devices—septic arthritis -   6. Preventive vaccination of general population     B) Passive and active vaccination, both with special attention to     T-cells with the latter one: It is an aim to induce a strong T     helper response during vaccination to achieve efficient humoral     response and also immunological memory. Up till now, there is no     direct evidence that T-cells play an important role in clearing S.     aureus infections, however, it was not adequately addressed, so far.     An effective humoral response against proteinaceous antigens must     involve T help, and is essential for developing memory. Naïve CD4+     cells can differentiated into Th1 or Th2 cells. Since, innate     immunological responses (cytokines) will influence this decision,     the involvement of T-cells might be different during an acute,     serious infection relative to immunization of healthy individuals     with subunit vaccines, not containing components which impair the     immune response during the natural course of the infection. The     consequences of inducing Th1 or Th2 responses are profound. Th1     cells lead to cell-mediated immunity, whereas Th2 cells provide     humoral immunity.     C) Preventive and Therapeutic Vaccines     Preventive: active vaccination/passive immunization of people in     high risk groups, before infection     Therapeutic: passive vaccination of the already sick. Active     vaccination to remove nasal carriage

Specific Example for an Application Elimination of MRSA Carriage and Prevention of Colonization of the Medical Staff

Carriage rates of S. aureus in the nares of people outside of the hospitals varies from 10 to 40%. Hospital patients and personnel have higher carriage rates. The rates are especially high in patients undergoing hemodialysis and in diabetics, drug addicts and patients with a variety of dermatologic conditions. Patients at highest risk for MRSA infection are those in large tertiary-care hospitals, particularly the elderly and immunocompromised, those in intensive care units, burn patients, those with surgical wounds, and patients with intravenous catheters.

The ELISA data strongly suggest that there is a pronounced IgA response to S. aureus, which is not obvious or known from the literature. Since the predominant mucosal immune response is the production of IgA with neutralizing activity, it is clear that the staphylococcal epitopes and antigens identified with the highly pure IgA preparations lead to an efficient mucosal vaccine.

-   -   Clear indication: Everybody's threat in the departments where         they perform operation (esp. orthopedics, traumatology, gen.         surgery)     -   Well-defined population for vaccination (doctors and nurses)     -   Health care workers identified as intranasal carriers of an         epidemic strain of S. aureus are currently treated with         mupirocin and rifampicin until they eliminate the bacteria.         Sometimes it is not effective, and takes time.     -   Available animal model: There are mice models for intranasal         carriage.

TABLE 1 ELISA titers of sera from non-infected individuals against multiple staphylococcal proteins. Sera BHI D1 + Map- ID# lysate LTA PG ClfA D3 FnBPA sdrE sdrC EBP enolase LP309 LP342 coagul Fib SrtA ClfB w 1 2 2 . . . 2 . . . 8 . . . 4 . . . 6 . . . 3 . . . 3 7 . . . 3 . . . 1 . . . 1 . . . 3 . . . 2 . . . 2 . . . 2 . . . 3 . . . 7 . . . 4 . . . 4 1****** 1****** 1 . . . 6 . . . 2 . . . 2 . . . 3 . . . 6 . . . 2 . . . 6, 7 . . . 8 . . . 3 . . . 4 . . . 1 . . . 3 . . . 5 6 4 . . . 5 . . . 7 6 . . . 7 . . . 7 . . . 5 . . . 4 . . . 7 . . . 8 8 . . . 8 . . . 5 . . . 7 . . . 8, 9 . . . 9 4, 5, 6 . . . 4 . . . 5 . . . 5 . . . 6 . . . 3 . . . 1 . . . 3, 4 . . . 1 . . . 5, 6 . . . 10 5, 6 . . . 11 6 . . . 6 . . . 12 4, 5 . . . 13 5 . . . 2 . . . 8 . . . 14 15 3 . . . 5 . . . 2, 5 . . . 6 . . . 7 . . . 8 . . . 8, 3 . . . 9 . . . 16 6 . . . 17 18 6, 1 . . . 3 . . . 4 . . . 7 . . . 19 1 . . . 20 21 2 . . . 2 . . . 22 23 4, 5, 6 . . . 5 . . . 3 . . . 6 . . . 2 . . . 7 . . . 4 . . . 6, 7 . . . 7 . . . 6, 7 . . . 2 . . . 2 . . . 24 4 . . . 6 . . . 8, 9 . . . 25 5 . . . 26 8 . . . 7 . . . 27 8 . . . 4 . . . 4, 5 . . . 4, 5 . . . 5 . . . 28 29 1 . . . 30 31 1 . . . 1 . . . 1 . . . 32 4 . . . 33 8 . . . 4 . . . 4 . . . 5 . . . 34 7, 2 . . . 2 . . . 1 . . . 6, 7 . . . 6 . . . 1 . . . 8 . . . 35 4, 5, 6 . . . 8 . . . 2, 5 . . . 1*** 3 . . . 4 . . . 3 . . . 36 3 . . . 37 7 . . . 7, 3 . . . 8 . . . 38 8 . . . 3, 4 . . . 39 40 7 . . . 6, 3 . . . 4, 5 . . . 8, 7 . . . 9 . . . Table I. ELISA Titers of Sera from Non-infected Individuals Against Multiple Staphylococcal Proteins.

Anti-staphylococcal antibody levels were measured individually by standard ELISA with total lysate prepared from S. aureus grown in BHI medium (BHI), lipoteichoic acid (LTA), peptidoglycan (PG), 13 recombinant proteins, representing cell surface and secreted proteins, such as clumping factor A and B (ClfA, ClfB), Fibronectin-binding protein (FnBPA), SD-repeat proteins (sdrC, sdrE), MHC Class II analogous protein (map-w), Elastin-binding protein (EBP), enolase (reported to be cell surface located and immunogenic), iron transport lipoproteins (LP309, LP342), sortase (srtA), coagulase (coa), extracellular fibrinogen-binding protein (fib). Two short synthetic peptides representing 2 of the five immunodominant D repeat domains from FnBPA was also included (D1+D3) as antigens. The individual sera were ranked based on the IgG titer, and obtained a score from 1-9. Score 1 labels the highest titer serum and score 8 or 9 labels the sera which were 8^(th) or 9^(th) among all the sera tested for the given antigen. It resulted in the analyses of the top 20 percentile of sera (8-9/40). The five “best sera” meaning the most hyper reactive in terms of anti-staphylococcal antibodies were selected based on the number of scores 1-8. **** means that the antibody reactivity against the particular antigen was exceptionally high (>2× ELISA units relative to the 2^(nd) most reactive serum).

TABLE 2a Immunogenic proteins identified by bacterial surface and ribosome display: S. aureus No. of Location of S. selected identified aureus Old Putative clones per immuno- Serum reactivity Seq ID no: antigenic ORF function ORF and genic with relevant region (DNA + protein number (by homology) predicted immunogenic aa** screen region (positive/total) Prot) SaA0003 ORF2963P repC 5-20, 37-44, 52-59, 87-94, 116-132 C: 3 aa 112-189 C: GSBYM94(112-189): 171, 172 26/30 SaA0003 ORF2967P repC 7-19, 46-57, 85-91, 110-117, 125-133, C: 18 aa 9-42 C: GSBYI53(9-42): 150, 158 140-149, 156-163, 198-204, aa 158-174 1/1 236-251, 269-275, 283-290, 318-323, 347-363 0093 ORF1879 SdrC 23-51, 75-80, 90-99, 101-107, 151-157, A: 1, D: 5, aa 98-182 A: GSBXL70(98-182): 34, 86 173-180, 186-205, 215-226, C: 1, F: 6, aa 684-764 9/30 239-263, 269-274, 284-304, 317-323, G: 2 aa 836-870 D: n.d. 329-336, 340-347, 360-366, C: GSBYH73(815-870): 372-379, 391-397, 399-406, 413-425, 3/16 430-436, 444-455, 499-505, 520-529, 553-568, 586-592, 600-617, 631-639, 664-678, 695-701, 891-903, 906-912, 926-940 0095 ORF1881 SdrE 25-45, 72-77, 147-155, 198-211, C: 12, aa 147-192 C: GSBYH31(147-192): 145, 153 217-223, 232-238, 246-261, 266-278, E: 2 2/14 281-294, 299-304, 332-340, E: GSBZA27(144-162): 353-360, 367-380, 384-396, 404-409, 23/41 418-429, 434-440, 448-460, 465-476, 493-509, 517-523, 531-540, 543-555, 561-566, 576-582, 584-591, 603-617, 633-643, 647-652, 668-674, 677-683, 696-704, 716-728, 744-752, 755-761, 789-796, 809-815, 826-840, 854-862, 887-903, 918-924, 1110-1116, 1125-1131, 1145-1159 0123 ORF1909 unknown 9-28, 43-48, 56-75, 109-126, 128-141, B: 3, E: 7, aa 168-181 B: GSBXF80(168-181): 35, 87 143-162, 164-195, 197-216, G: 1 5/27 234-242, 244-251 E: GSBZC17(168-181): 25/41 0160 ORF1941 unknown 4-10, 20-42, 50-86, 88-98, 102-171, A: 1 aa 112-188 A: GSBXO07(112-188): 36, 88 176-182, 189-221, 223-244, 246-268, 5/30 276-284, 296-329 0222 ORF1988 homology with 4-9, 13-24, 26-34, 37-43, 45-51, A: 52, aa 45-105 A: GSBXM63(65-95): 37, 89 ORF1 59-73, 90-96, 99-113, 160-173, C: 18*, aa 103-166 1/1 178-184, 218-228, 233-238, 255-262 H: 19 aa 66-153 A: GSBXM82(103-166): 14/29 A: GSBXK44- bmd3(65-153): 47/51 0308 ORF2077 Complement, 13-27, 42-63, 107-191, 198-215, A: 6, B: 2, complement A: GSBXK03(bp473- 38, 90 unknown 218-225, 233-250 C: 47, bp 474-367 367): 28/69 E: 35 B: GSBXD29(bp465- 431): 10/27 0317 ORF2088 preprotein 16-29, 64-77, 87-93, 95-101, 127-143, A: 1 aa 1-19 A: GSBXP37(1-19): 39, 91 translocase 150-161, 204-221, 225-230, 6/29 seca subunit 236-249, 263-269, 281-309, 311-325, 337-343, 411-418, 421-432, 435-448, 461-467, 474-480, 483-489, 508-516, 542-550, 580-589, 602-611, 630-636, 658-672, 688-705, 717-723, 738-746, 775-786, 800-805, 812-821, 828-834 0337 ORF2110 Hypothetical 26-53, 95-123, 164-176, 189-199 D: 12 aa 8-48 D: n.d. 40, 92 protein 0358 ORF2132 Clumping 8-35, 41-48, 59-66, 87-93, 139-144, C: 1, D: 2, aa 706-809 D: n.d. 41, 93 factor A 156-163, 198-209, 215-229, 236-244, E: 1 246-273, 276-283, 285-326, 328-342, 349-355, 362-370, 372-384, 396-402, 405-415, 423-428, 432-452, 458-465, 471-477, 484-494, 502-515, 540-547, 554-559, 869-875, 893-898, 907-924 0360 ORF2135 extracellular 7-13, 15-23, 26-33, 68-81, 84-90, A: 46, aa 22-56 A: GSBXK24(23-55): 42, 94 Empbp matrix and 106-117, 129-137, 140-159, 165-172, B: 21, aa 23-99 1/1 plasma 177-230, 234-240, 258-278, C: 11, aa 97-115 B: GSBXB43(39-54): binding protein 295-319 E: 2, aa 233-250 58/71 F: 18, aa 245-265 A: GSBXK02- G: 7, bmd1(22-99): 59/59 H: 12 B: GSBXD82- bdb19(97-115): 1/1 F: SALAL03(233-250): 15/41 0453 ORF2227 coma operon 17-25, 27-55, 84-90, 95-101, 115-121 C: 3 aa 55-101 C: GSBYG07(55-101): 146, 154 protein 2 1/1 0569 ORF1640 V8 protease 5-32, 66-72, 87-98, 104-112, 116-124, A: 1, F: 1 aa 174-249 A: GSBXS51(174-249): 32, 84 128-137, 162-168, 174-183, 11/30 248-254, 261-266, 289-303, 312-331 0576 ORF1633 autolysin, 4-19, 57-70, 79-88, 126-132, 144-159, A: 21, aa 6-66 A: GSBXN93(6-66): 31, 83 Autolysin adhesion 161-167, 180-198, 200-212, B: 46, aa 65-124 5/16 233-240, 248-255, 276-286, 298-304, C: 55, aa 579-592 C: GSBYH05(45-144): 309-323, 332-346, 357-366, E: 5, aa 590-604 7/8 374-391, 394-406, 450-456, 466-473, F: 85, A: GSBXK66- 479-487, 498-505, 507-519, H: 19 bmd18(65-124): 521-530, 532-540, 555-565, 571-581, 16/30 600-611, 619-625, 634-642, B: GSBXB89(108-123): 650-656, 658-665, 676-682, 690-699, 1/1 724-733, 740-771, 774-784, B: GSBXB02(590-603): 791-797, 808-815, 821-828, 832-838, 39/71 876-881, 893-906, 922-929, F: SALAM15(579-592): 938-943, 948-953, 969-976, 1002-1008, 25/41 1015-1035, 1056-1069, 1105-1116, 1124-1135, 1144-1151, 1173-1181, 1186-1191, 1206-1215, 1225-1230, 1235-1242 0657 ORF LPXTGVI 9-33, 56-62, 75-84, 99-105, 122-127, A: 2, aa 527-544 B: GSBXE07- 1/165, unknown protein 163-180, 186-192, 206-228, B: 27, bdb1(527-542): 142 233-240, 254-262, 275-283, 289-296, F: 15 11/71 322-330, 348-355, 416-424, F: SALAX70(526-544): 426-438, 441-452, 484-491, 541-549, 11/41 563-569, 578-584, 624-641 0749 ORF1462 Carbamoyl- 8-23, 31-38, 42-49, 61-77, 83-90, C: 2 aa 630-700 C: GSBYK17(630-700): 144, 152 phosphate 99-108, 110-119, 140-147, 149-155, 5/9 synthase 159-171, 180-185, 189-209, 228-234, 245-262, 264-275, 280-302, 304-330, 343-360, 391-409, 432-437, 454-463, 467-474, 478-485, 515-528, 532-539, 553-567, 569-581, 586-592, 605-612, 627-635, 639-656, 671-682, 700-714, 731-747, 754-770, 775-791, 797-834, 838-848, 872-891, 927-933, 935-942, 948-968, 976-986, 1000-1007, 1029-1037 944 ORF1414 Yfix 6-33, 40-46, 51-59, 61-77, 84-104, D: 4 aa 483-511 D: n.d. 30, 82 112-118, 124-187, 194-248, 252-296, 308-325, 327-361, 367-393, 396-437, 452-479, 484-520, 535-545, 558-574, 582-614, 627-633, 656-663, 671-678, 698-704, 713-722, 725-742, 744-755, 770-784, 786-800, 816-822, 827-837 1050 ORF1307 unknown 49-72, 76-83, 95-105, 135-146, A: 1, aa 57-128 A: GSBXM26(57-128): 28, 80 148-164, 183-205 H: 45 7/30 1209 ORF3006 hemN homolog 12-36, 43-50, 58-65, 73-78, 80-87, B: 7, F: 8 aa 167-181 B: GSBXB76(167-179):  54, 106 108-139, 147-153, 159-172, 190-203, 25/71 211-216, 224-232, 234-246, F:SALBC54(169-183): 256-261, 273-279, 286-293, 299-306, 18/41 340-346, 354-366 1344 ORF0212 NifS protein 8-16, 22-35, 49-58, 70-77, 101-121, A: 11 aa 34-94 A: GSBXK59-  5, 141 homolog 123-132, 147-161, 163-192, 203-209, bmd21(34-94): 6/29 216-234, 238-249, 268-274, 280-293, 298-318, 328-333, 339-345, 355-361, 372-381 1356 ORF0197 Hypothetical 28-55, 82-100, 105-111, 125-131, D: 12 aa 1-49 D: n.d.  4, 57 protease 137-143 1361 ORF0190 LPXTGV 5-39, 111-117, 125-132, 134-141, A: 1, aa 37-49 B: GSBXF81(37-49):  3, 56 protein 167-191, 196-202, 214-232, 236-241, B: 23, aa 63-77 1/1 244-249, 292-297, 319-328, E: 3, aa 274-334 B: GSBXD45- 336-341, 365-380, 385-391, 407-416, F: 31 bdb4(62-77): 12/70 420-429, 435-441, 452-461, A: GSBXL77(274-334): 477-488, 491-498, 518-532, 545-556, 5/30 569-576, 581-587, 595-602, F: SALAP81(62-77): 604-609, 617-640, 643-651, 702-715, 10/41 723-731, 786-793, 805-811, 826-839, 874-889 1371 ORF0175 YtpT, 37-42, 57-62, 121-135, 139-145, C: 3, E: 2, aa 624-684 C: GSBYG95(624-684): 143, 151 conserved 183-190, 204-212, 220-227, 242-248, G: 1 aa 891-905 7/22 hypothetical 278-288, 295-30, 304-309, E: GSBZB45(891-905): protein 335-341, 396-404, 412-433, 443-449, 10/41 497-503, 505-513, 539-545, 552-558, 601-617, 629-649, 702-711, 736-745, 793-804, 814-829, 843-858, 864-885, 889-895, 905-913, 919-929, 937-943, 957-965, 970-986, 990-1030, 1038-1049, 1063-1072, 1080-1091, 1093-1116, 1126-1136, 1145-1157, 1163-1171, 1177-1183, 1189-1196, 1211-1218, 1225-1235, 1242-1256, 1261-1269 1491 ORF0053 Cmp binding 12-29, 34-40, 63-71, 101-110, 114-122, A: 7, C: 2, aa 39-94 A: GSBXM13(39-94): 2/166, factor 1 130-138, 140-195, 197-209, E: 7, F: 4 10/29 55 homolog 215-229, 239-253, 255-274 F: SALAY30(39-53): 4/41 1616 ORF1180 leukocidin F 16-24, 32-39, 43-49, 64-71, 93-99, A: 10 aa 158-220 A: GSBXK06(158-220): 27, 79 homolog 126-141, 144-156, 210-218, 226-233, 8/29 265-273, 276-284 1618 ORF1178 LukM homolog 5-24, 88-94, 102-113, 132-143, A: 13, aa 31-61 A: GSBXK60(31-61): 26, 78 163-173, 216-224, 254-269, 273-278, B: 3 aa 58-74 20/29 305-313, 321-327, 334-341 C: 36, B: GSBXB48(58-74): E: 4, 49/71 F: 12, F: SALAY41(58-74): G: 2, 30/41 H: 10 1632 ORF1163 SdrH homolog 7-35, 54-59, 247-261, 263-272, B: 6, aa 105-119 B: GSBXG53(168-186): 25, 77 302-320, 330-339, 368-374, 382-411 E: 11, aa 126-143 39/71 F: 34 aa 168-186 F: SALAP07(105-119): 11/41 1763 ORF1024 unknown 5-32, 35-48, 55-76 C: 3 complement C: GSBY130(98aa): 1/1 24, 76 bp 237-170 1845 ORF0942 Hyaluronate 10-26, 31-44, 60-66, 99-104, 146-153, D: 5, F: 2 aa 208-224 D: n.d. 23, 75 lyase 163-169, 197-205, 216-223, aa 672-727 226-238, 241-258, 271-280, 295-315, 346-351, 371-385, 396-407, 440-446, 452-457, 460-466, 492-510, 537-543, 546-551, 565-582, 590-595, 635-650, 672-678, 686-701, 705-712, 714-721, 725-731, 762-768, 800-805 1951 ORF0831 homology with 5-22, 42-50, 74-81, 139-145, 167-178, A: 223, aa: 137-237 B: GSBXC07(180-190): 22, 74 ORF1 220-230, 246-253, 255-264 B: 56, aa 250-267 1/1 C: 167, A: GSBXK29(177-195): E: 43, 15/29 F: 100, B: GSBXD43(250-267): G: 13, 10/71 H: 102 F: SALAM13(178-191): 20/41 1955 ORF0826 homology with 4-9, 15-26, 65-76, 108-115, 119-128, A: 1, B: 3, aa 38-52. A: GSBXR10(66-114): 21, 73 ORF1 144-153 E: 1, F: 8 aa 66-114 5/30 F: SALAM67(37-52): 16/41 2031 ORF0749 unknown 10-26, 31-43, 46-58, 61-66, 69-79, B: 2, F: 2 aa 59-74 B: GSBXC01(59-71): 20, 72 85-92, 100-115, 120-126, 128-135, 11/26 149-155, 167-173, 178-187, 189-196 202-222, 225-231, 233-240, 245-251, 257-263, 271-292, 314-322, 325-334, 339-345 2086 ORF0691 IgG binding 6-20, 53-63, 83-90, 135-146, 195-208, A: 1, B: 8, aa 208-287 A: GSBXS55(208-287): 19, 71 Sbi protein 244-259, 263-314, 319-327, E: 24, aa 261-276 38/46 337-349, 353-362, 365-374, 380-390, F: 9, aa 286-314 B: GSBXB34(299-314):: 397-405, 407-415 G: 137 11/71 F: SALAX32(261-276): 21/41 2180 ORF0594 LPXTGIV 11-20, 26-47, 69-75, 84-92, 102-109, A: 3, C: 3, aa 493-587 A: GSBXS61(493-555): 18, 70 protein 119-136, 139-147, 160-170, E: 6, F: 2, aa 633-715 1/1 178-185, 190-196, 208-215, 225-233, H: 6 aa 704-760^(#) A: GSBXL64(496-585): 245-250, 265-272, 277-284, aa 760-832 1/1 300-306, 346-357, 373-379, 384-390, (aa 832- A: GSBXS92(760-841): 429-435, 471-481, 502-507, 887)^(#) 1/1 536-561, 663-688, 791-816, 905-910, A: bmd4(704-760): 919-933, 977-985, 1001-1010, 16/30^(#) 1052-1057, 1070-1077, 1082-1087, (A: bmd4(830-885): 1094-1112 16/30)^(#) F: SALBC43(519-533): 4/41 2184 ORF0590 FnbpB 5-12, 18-37, 104-124, 139-145, A: 2, C: 4, aa 701-777 A: GSBXM62(702-777): 17, 69 154-166, 175-181, 185-190, 193-199, G: 9 aa 783-822 28/28 203-209, 235-244, 268-274, A: GSBXR22(783-855): 278-292, 299-307, 309-320, 356-364, 1/1 375-384, 390-404, 430-440, 450-461, 488-495, 505-511, 527-535, 551-556, 567-573, 587-593, 599-609, 624-631, 651-656, 665-671, 714-726, 754-766, 799-804, 818-825, 827-833, 841-847, 855-861, 876-893, 895-903, 927-940 2186 ORF0588 Fnbp 8-29, 96-105, 114-121, 123-129, A: 4, C: 4, aa 710-787 C: GSBYN05(710-787): 16, 68 141-147, 151-165, 171-183, 198-206, D: 5, E: 2 aa 855-975 19/25 222-232, 253-265, 267-277, aa 916-983 D: n.d. 294-300, 302-312, 332-338, 362-368, A: GSBXP01(916-983): 377-383, 396-402, 410-416, 17/30 451-459, 473-489, 497-503, 537-543, 549-559, 581-600, 623-629, 643-649, 655-666, 680-687, 694-700, 707-712, 721-727, 770-782, 810-822, 874-881, 883-889, 897-903, 911-917, 925-931, 933-939, 946-963, 965-973, 997-1010 2224 ORF0551 unknown 49-56, 62-68, 83-89, 92-98, 109-115, B: 2 aa 34-46 B: GSBXD89(34-46): 15, 67 124-131, 142-159, 161-167, 1/1 169-175, 177-188, 196-224, 230-243, 246-252 2254 ORF0519 Conserved 14-22, 32-40, 52-58, 61-77, 81-93, D: 3 aa 403-462 D: n.d. 14, 66 hypothetical 111-117, 124-138, 151-190, 193-214, protein 224-244, 253-277, 287-295, 307-324, 326-332, 348-355, 357-362, 384-394, 397-434, 437-460, 489-496, 503-510, 516-522, 528-539, 541-547, 552-558, 563-573, 589-595, 602-624, 626-632, 651-667, 673-689, 694-706, 712-739, 756-790 2264 ORF0509 ORF1; 5-31, 47-55, 99-104, 133-139, 156-172, A: 131, aa 7-87 A: GSBXP22(145-196): 13, 65 homology with 214-224, 240-247 B: 51, aa 133-242 1/1 putative C: 13, A: GSBXK05- secreted E: 43, bmd16(178-218): antigen F: 78, 6/29 precursor from G: 2, B: GSBXE24- S. epidermidis H: 17 bdb20(167-178):1/1 F: SALAQ91(173-184): 15/41 2268 ORF0503 IsaA, possibly 7-19, 26-45, 60-68, 94-100, 111-119, A: 7, aa 67-116 A: GSBXK88(67-116): 12, 64 adhesion/ 126-137, 143-148, 169-181, B: 65, aa 98-184 1/1 aggregation 217-228 C: 3, E: 2, aa 182-225 A: GSBXN19(98-184): F: 53 22/29 A: GSBXN32(182-225): 34/71 B: GSBXB71(196-209): 16/29 F: SALAL22(196-210): 16/41 2344 ORF0426 Clumping 4-10, 17-45, 120-127, 135-141, D: 9, E: 1, aa 706-762 D: n.d. 11, 63 factor B 168-180, 187-208, 216-224, 244-254, F: 3, H: 4 aa 810-852 256-264, 290-312, 322-330, 356-366, 374-384, 391-414, 421-428, 430-437, 442-449, 455-461, 464-479, 483-492, 501-512, 548-555, 862-868, 871-876, 891-904 2351 ORF0418 aureolysin 10-29, 46-56, 63-74, 83-105, 107-114, A: 1, C: 6 aa 83-156 A: GSBXO46(83-156): 10, 62 138-145, 170-184, 186-193, 14/29 216-221, 242-248, 277-289, 303-311, 346-360, 379-389, 422-428, 446-453, 459-469, 479-489, 496-501 2359 ORF0409 ISSP, 4-29, 92-99, 119-130, 228-236, B: 4, F: 11 aa 168-184 B: GSBXD01(168-184):  9, 61 immunogenic 264-269, 271-280, 311-317, 321-331, aa 206-220 1/1 secreted 341-353, 357-363, 366-372, aa 297-309 B: GSBXD62(205-220): protein 377-384, 390-396, 409-415, 440-448, 1/1 precursor, 458-470, 504-520, 544-563, B: GSBXC17(297-309): putative 568-581, 584-592, 594-603, 610-616 6/27 F: SALAL04(205-220): 9/41 2378 ORF0398 SrpA 18-23, 42-55, 69-77, 85-98, 129-136, C: 1, D: 7, aa 198-258 C: GSBYI73(646-727):  8, 60 182-188, 214-220, 229-235, F: 4, aa 646-727 2/9 242-248, 251-258, 281-292, 309-316, H: 11 aa 846-857 F: SALAO33(846-857): 333-343, 348-354, 361-367, aa 2104- 10/41 393-407, 441-447, 481-488, 493-505, 2206 D: n.d. 510-515, 517-527, 530-535, 540-549, 564-583, 593-599, 608-621, 636-645, 656-670, 674-687, 697-708, 726-734, 755-760, 765-772, 785-792, 798-815, 819-824, 826-838, 846-852, 889-904, 907-913, 932-939, 956-964, 982-1000, 1008-1015, 1017-1024, 1028-1034, 1059-1065, 1078-1084, 1122-1129, 1134-1143, 1180-1186, 1188-1194, 1205-1215, 1224-1230, 1276-1283, 1333-1339, 1377-1382, 1415-1421, 1448-1459, 1467-1472, 1537-1545, 1556-1566, 1647-1654, 1666-1675, 1683-1689, 1722-1737, 1740-1754, 1756-1762, 1764-1773, 1775-1783, 1800-1809, 1811-1819, 1839-1851, 1859-1866, 1876-1882, 1930-1939, 1947-1954, 1978-1985, 1999-2007, 2015-2029, 2080-2086, 2094-2100, 2112-2118, 2196-2205, 2232-2243 2466 ORF0302 YycH protein 16-38, 71-77, 87-94, 105-112, 124-144, D: 14 aa 401-494 D: n.d.  7, 59 158-164, 169-177, 180-186, 194-204, 221-228, 236-245, 250-267, 336-343, 363-378, 385-394, 406-412, 423-440, 443-449 2470 ORF0299 Conserved 4-9, 17-41, 50-56, 63-69, 82-87, C: 3 aa 414-455 C: GSBYH60(414-455): 169, 170 hypothetical 108-115, 145-151, 207-214, 244-249, 28/31 protein 284-290, 308-316, 323-338, 348-358, 361-378, 410-419, 445-451, 512-522, 527-533, 540-546, 553-558, 561-575, 601-608, 632-644, 656-667, 701-713, 727-733, 766-780 2498 ORF0267 Conserved 33-43, 45-51, 57-63, 65-72, 80-96, D: 12 aa 358-411 D: 17/21  6, 58 hypothetical 99-110, 123-129, 161-171, 173-179, aa 588-606 protein 185-191, 193-200, 208-224, 227-246, 252-258, 294-308, 321-329, 344-352, 691-707 2548 ORF2711 IgG binding 4-16, 24-57, 65-73, 85-91, 95-102, A: 55, aa 1-48 A: GSBXK68(1-73):  53, 105 protein A 125-132, 146-152, 156-163, 184-190, B: 54, aa 47-143 21/30 204-210, 214-221, 242-252, C: 35, aa 219-285 A: GSBXK41(47-135): 262-268, 272-279, 300-311, 320-337, F: 59, aa 345-424 1/1 433-440, 472-480, 505-523 G: 56, A: GSBXN38(219-285): H: 38 19/30 A: GSBXL11(322-375): 10/30 B: GSBXB22(406-418): 37/71 F: SALAM17(406-418): 29/41 2577 ORF2683 Hypothetical 4-21, 49-56, 65-74, 95-112, 202-208, C: 6 aa 99-171 C: GSBYL56(99-171): 149, 157 protein 214-235 1/1 2642 ORF2614 unknown 34-58, 63-69, 74-86, 92-101, 130-138, C: 1, E: 1 aa 5-48 C: bhe3(5-48):  52, 104 142-150, 158-191, 199-207, 25/30^(##) 210-221, 234-249, 252-271 2664 ORF2593 Conserved 7-37, 56-71, 74-150, 155-162, 183-203, D: 35 aa 77-128 D: n.d.  51, 103 hypothetical 211-222, 224-234, 242-272 protein 2670 ORF2588 Hexose 18-28, 36-49, 56-62, 67-84, 86-95, D: 16 aa 328-394 D: n.d.  50, 102 transporter 102-153, 180-195, 198-218, 254-280 284-296, 301-325, 327-348, 353-390, 397-402, 407-414, 431-455 2680 ORF2577 Coagulase 4-18, 25-31, 35-40, 53-69, 89-102, C: 26, aa 438-516 C: GSBYH16(438-516): 148, 156 147-154, 159-165, 185-202, 215-223, G: 4, H: 8 aa 505-570 3/5 284-289, 315-322, 350-363, aa 569-619 C: GSBYG24(505-570): 384-392, 447-453, 473-479, 517-523, 1/7 544-550, 572-577, 598-604, C: GSBYL82(569-619): 617-623 2/7 2740 ORF2515 Hypothetical 5-44, 47-55, 62-68, 70-78, 93-100, D: 4 aa 1-59 D: n.d.  49, 101 protein 128-151, 166-171, 176-308 2746 ORF2507 homology with 5-12, 15-20, 43-49, 94-106, 110-116, A: 1, aa 63-126 A: GSBXO40(66-123):  48, 100 ORF1 119-128, 153-163, 175-180, H: 13 8/29 185-191, 198-209, 244-252, 254-264, 266-273, 280-288, 290-297 2797 ORF2470 unknown 10-27, 37-56, 64-99, 106-119, 121-136, B: 3, E: 2, aa 183-200 B: GSBXE85(183-200): 47, 99 139-145, 148-178, 190-216, F: 13, aa 349-363 11/27 225-249, 251-276, 292-297, 312-321, H: 3 F: SALAQ47(183-200): 332-399, 403-458 8/41 2798 ORF2469 Lipase (geh) 12-35, 93-99, 166-179, 217-227, A: 41, aa 48-136 C: GSBYG01(48-136): 46, 98 239-248, 269-276, 288-294, 296-320, B: 42, aa 128-172 2/6 322-327, 334-339, 344-356, C: 3, aa 201-258 A: GSBXM31- 362-371, 375-384, 404-411, 433-438, F: 35, bmd12(128-188): 443-448, 455-464, 480-486, G: 1, 11/30 497-503, 516-525, 535-541, 561-570, H: 11 B: GSBXE16(165-177): 579-585, 603-622, 633-641 10/30 A: GSBXN20(201-258): 8/30 F: SALAW05(165-177): 13/41 2815 ORF2451 Conserved 5-32, 34-49 D: 21 aa 1-43 D: n.d. 45, 97 hypothetical protein 2914 ORF2351 metC 39-44, 46-80, 92-98, 105-113, 118-123, A: 1, aa 386-402 A: GSBXM18(386-402): 44, 96 133-165, 176-208, 226-238, C: 14, 17/29 240-255, 279-285, 298-330, 338-345 F: 2 350-357, 365-372, 397-402, 409-415, 465-473, 488-515, 517-535, 542-550, 554-590, 593-601, 603-620, 627-653, 660-665, 674-687, 698-718, 726-739 2960 ORF2298 putative 13-36, 40-49, 111-118, 134-140, C: 101, aa 1-85 C: GSBYG32(1-85):: 43, 95 Exotoxin 159-164, 173-183, 208-220, 232-241, E: 2, aa 54-121 6/7 245-254, 262-271, 280-286, H: 58 aa 103-195 C: GSBYG61- 295-301, 303-310, 319-324, 332-339 bhe2(54-121): 26/30 C: GSBYN80(103-195): 13/17 2963 ORF2295 putative 13-28, 40-46, 69-75, 86-92, 114-120, C: 3, E: 3, aa 22-100 C: GSBYJ58(22-100): 147, 155 Exotoxin 126-137, 155-172, 182-193, G: 1 9/15 199-206, 213-221, 232-238, 243-253 270-276, 284-290 3002 ORF1704 homology with 4-21, 28-40, 45-52, 59-71, 92-107, A: 2, C: 1, aa 21-118 A: GSBXL06(21-118): 33, 85 ORF1 123-137, 159-174, 190-202, 220-229, H: 4 50/52 232-241, 282-296, 302-308, 312-331 3200 ORF1331 putative 6-15, 22-32, 58-73, 82-88, 97-109, A: 11, aa 5-134 A: GSBXL07(5-134): 29, 81 extracellular 120-131, 134-140, 151-163, 179-185, B: 11, 6/28 matrix binding 219-230, 242-255, 271-277, C: 36 protein 288-293, 305-319, 345-356, 368-381, 397-406, 408-420, 427-437, 448-454, 473-482, 498-505, 529-535, 550-563, 573-580, 582-590, 600-605, 618-627, 677-685, 718-725, 729-735, 744-759, 773-784, 789-794, 820-837, 902-908, 916-921, 929-935, 949-955, 1001-1008, 1026-1032, 1074-1083, 1088-1094, 1108-1117, 1137-1142, 1159-1177, 1183-1194, 1214-1220, 1236-1252, 1261-1269, 1289-1294, 1311-1329, 1336-1341, 1406-1413, 1419-1432, 1437-1457, 1464-1503, 1519-1525, 1531-1537, 1539-1557, 1560-1567, 1611-1618, 1620-1629, 1697-1704, 1712-1719, 1726-1736, 1781-1786, 1797-1817, 1848-1854, 1879-1890, 1919-1925, 1946-1953, 1974-1979 Bacterial surface display: A, LSA250/1 library in fhuA with patient sera 1 (655); B, LSA50/6 library in lamB with patient sera 1 (484); C, LSA250/1 library in fhuA with IC sera 1 (571); E, LSA50/6 library in lamB with IC sera 2 (454); F, LSA50/6 library in lamB with patient sera P1 (1105); G, LSA50/6 library in lamb with IC sera 1 (471) ); H, LSA250/1 library in fhuA with patient sera 1 (IgA, 708). Ribosome display: D, LSA250/1 library with IC sera (1686). *identified 18 times of 33 screened; was therefore eliminated from screen C. **prediction of antigenic sequences longer than 5 amino acids was performed with the programme ANTIGENIC (Kolaskar and Tongaonkar, 1990); ^(#)identical sequence present twice in ORF; ^(##)clone not in database (not sequence by TIGR).

TABLE 2b Additional immunogenic proteins identified by bacterial surface and ribosome display: S. aureus No. of selected Location of S. clones identified Seq ID aureus per ORF immuno- no: antigenic Putative function and genic Serum reactivity with relevant (DNA + protein (by homology) predicted immunogenic aa** screen region region (positive/total) Prot) ARF0280 Putative protein 7-14 F: 6 aa 25-43 SALAM59(25-43): 1/1 401, 402 CRF0145 Putative protein 18-28, 31-37, 40-47, 51-83, 86-126 F: 5 aa 81-90 SALAZ40(81-90): 2/12 403, 404 CRF0250 Putative protein 4-24, 26-46, 49-86 G: 8 aa 60-76 SALAJ87(60-76): n.d. 365, 378 CRF0308 Putative protein 40-46 A: 6, B: 2, aa 5-38 A: GSBXK03(7-36): 28/69 391, 392 C: 47, B: GSBXD29(10-20): 10/27 E: 35 CRF0337 Unknown 4-17 D: 3 aa 1-20 D: n.d. 469; 486 CRF0497 Putative protein 4-28, 31-53, 58-64 B: 13, F: 5 aa 18-34 GSBXF31(19-34): 1/7 366, 379 CRF0538 Unknown 4-20 D: 7 aa 1-11 D: n.d. 470; 487 CRF0750 Putative protein 4-11, 18-24, 35-40 G: 44 aa 25-39 SALAG92(26-39): n.d. 367, 380 CRF1145 Unknown 4-57 D: 28 aa 16-32 D: n.d. 464; 481 CRF1247 Putative protein 4-25, 27-56 F: 6 aa 36-46 SALAR23(36-46): n.d. 368, 381 CRF1256 Putative protein 19-25, 38-47, 55-74, 77-87 G: 5 aa 54-67 SALAG65(54-67): n.d. 369, 382 CRF1356 Unknown 8-15; 18-24; 27-38 D: 5 aa 5-33 D: n.d. 471; 488 CRF1763 Putative protein 4-9, 23-41, 43-58, 71-85 C: 3 aa 1-22 C: GSBYI30(1-22): 1/1 407, 408 CRF1783 Unknown 8-161 D: 5 aa 76-127 D: n.d. 465; 482 CRF1845 Unknown 4-28; 30-36 D: 272 aa 1-17 D: n.d. 472; 489 CRF1861 Unknown 6-11; 13-34; 36-50 D: 8 aa 4-27 D: n.d. 466; 483 CRF1928 Putative protein 4-9, 17-30 F: 9 aa 13-22 SALAR41(13-22): n.d. 370, 383 CRF2004 Putative protein 18-38 F: 13 aa 16-32 SALAM75(16-32): n.d. 371, 384 CRF2155 Putative protein 4-15, 30-58 F: 9 aa 54-66 SALAQ54(54-66): 1/12 372, 385 CRF2180 Putative protein 4-61, 65-72, 79-95, 97-106 E: 13 aa 86-99 GSBZE08(86-99): n.d. 373, 386 CRF2207 Unknown 4-13 D: 3 aa 17-39 D: n.d. 473; 490 CRF2305 Putative protein 4-9, 22-33, 44-60 C: 5 aa 80-116 GSBYL75(80-116): n.d. 374, 387 CRF2341 Putative protein 4-23, 30-44, 49-70 F: 8 aa 46-55 SALAW31(46-55): n.d. 375, 388 CRF2349 Putative protein 4-32, 39-46, 62-69, 77-83 B: 10, F: 4 aa 46-67 GSBXC92(52-67): 2/11 376, 389 CRF2356 Unknown 4-18 D: 3 aa 3-18 D: n.d. 475; 492 CRF2452 Unknown 4-31 D: 9 aa 7-21 D: n.d. 476; 493 CRF2498 Putative protein 4-29, 31-41 G: 8 aa 2-15 SALAF30(3-15): n.d. 377, 390 CRF2553 Unknown 4-35; 37-42 D: 4 aa 1-20 D: n.d. 474; 491 CRF2578 Unknown 5-25; 30-39 D: 11 aa 9-30 D: n.d. 467; 484 CRF2664 Unknown 11-21 D: 17 aa 1-14 D: n.d. 477; 494 CRF2729 Putative protein 10-41, 50-57 F: 3 aa 40-56 SALAQ25(40-56): 1/1 405, 406 CRF2863/1 Unknown 4-43 D: 78 aa 17-40 D: n.d. 478; 495 CRF2863/2 Unknown 4-46 D: 78 aa 44-49 D: n.d. 479; 496 CRFA002 Unknown 17-39; 52-59 D: 3 aa 38-55 D: n.d. 463; 480 CRFN1 Unknown 5-20; 37-44; 52-59; 87-94; 116-132 D: 4 aa 94-116 D: n.d. 468; 485 ORF0188 UDP-N-acetyl- 11-18, 43-56, 58-97, 100-118, 120-148, B: 4, F: 29 aa 197-210 SALAM14(198-209): n.d. 397, 398, D-mannosamine 152-171, 195-203, 207-214, transferase, 220-227, 233-244 putative ORF0254 Multidrug efflux 4-33, 35-56, 66-99, 109-124, 136-144 D: 3 aa 155-175 D: n.d. 297, 325 transporter 151-180, 188-198, 201-236, 238-244, 250-260, 266-290, 294-306, 342-377 ORF0307 Conserved 4-23, 25-67, 76-107, 109-148 D: 3 aa 9-44 D: n.d. 298, 326 hypothetical protein ORF0452 Conserved 4-35, 41-47, 55-75, 77-89, 98-113, D: 5 aa 105-122 D: n.d. 299, 327 hypothetical 116-140, 144-179, 194-215, 232-254, protein 260-273, 280-288, 290-302, 315-323, 330-369, 372-385, 413-432 ORF0456 Na+/ 4-81 D: 66 aa 1-21 D: n.d. 300, 328 H+Antiporter ORF0556 Iron(III)dicitrate 5-23, 50-74, 92-99, 107-122, 126-142, D: 10 aa 1-18 D: n.d. 301, 329 binding protein 152-159, 172-179, 188-196, 211-218, 271-282 ORF0629 Hypothetical 9-44, 63-69, 75-82, 86-106, 108-146, D: 313 aa 13-37 D: n.d. 302, 330 Protein 153-161, 166-178, 185-192, 233-239, 258-266, 302-307 ORF0637 GTP-binding 10-19, 22-32, 95-105, 112-119, 121-133 F: 3 aa 107-119 F: SALAX70(107-119): 10/41 393, 395 protein TypA 140-154, 162-174, 186-200, 207-224, 238-247, 254-266, 274-280, 288-294, 296-305, 343-351, 358-364, 366-373, 382-393, 403-413, 415-422, 440-447, 499-507, 565-575, 578-588 ORF0713 Conserved 22-51, 53-71, 80-85, 93-99, 105-112, D: 3 aa 487-513 D: n.d. 303, 331 hypothetical 123-146, 151-157, 165-222, transmembrane 226-236, 247-270, 290-296, 301-324, protein, 330-348, 362-382, 384-391, putative 396-461, 463-482, 490-515 ORF0788 Cell division 104-111, 158-171, 186-197, 204-209, D: 4 aa 152-178 D: n.d. 304, 332 protein 230-247, 253-259, 269-277, 290-314, 330-340, 347-367, 378-388 ORF0797 Conserved 11-40, 56-75, 83-102, 112-117, 129-147, D: 12 aa 196-218 D: n.d. 305, 333 hypothetical 154-168, 174-191, 196-270, protein 280-344, 354-377, 380-429, 431-450, 458-483, 502-520, 525-532, 595-602, 662-669, 675-686, 696-702, 704-711, 720-735, 739-748, 750-756, 770-779, 793-800, 813-822, 834-862 ORF0836 Cell Division 34-91, 100-119, 126-143, 147-185, D: 5 aa 26-56 D: n.d. 306, 334 Protein 187-197, 319-335, 349-355, 363-395, 397-412, 414-422, 424-440, 458-465, 467-475, 480-505, 507-529, 531-542, 548-553, 577-589, 614-632, 640-649, 685-704, 730-741, 744-751, 780-786 ORP1318 Amino acid 11-21, 25-32, 34-54, 81-88, 93-99, D: 8 aa 127-152 D: n.d. 307, 335 permease 105-117, 122-145, 148-174, 187-193, 203-218, 226-260, 265-298, 306-318, 325-381, 393-399, 402-421, 426-448 ORF1321 Pyruvat kinase 4-11, 50-67, 89-95, 103-109, 112-135, E: 6 aa 420-432 E: GSBZE16(420-432): 5/41 197, 216 139-147, 158-170, 185-204, 213-219, 229-242, 248-277, 294-300, 316-323, 330-335, 339-379, 390-402, 408-422, 431-439, 446-457, 469-474, 484-500, 506-513, 517-530, 538-546, 548-561 ORF1388 LPXTG cell wall 11-31, 86-91, 103-111, 175-182, D: 3 aa 508-523 D: n.d. 308, 336 anchor motif 205-212, 218-226, 242-247, 260-269, 279-288, 304-313, 329-334, 355-360, 378-387, 390-399, 407-435, 468-486, 510-516, 535-547, 574-581, 604-615, 635-646, 653-659, 689-696, 730-737, 802-812, 879-891, 893-906, 922-931, 954-964, 997-1009, 1031-1042, 1089-1096, 1107-1120, 1123-1130, 1149-1162, 1176-1184, 1192-1207, 1209-1215, 1253-1259, 1265-1275, 1282-1295, 1304-1310, 1345-1361, 1382-1388, 1394-1400, 1412-1430, 1457-1462, 1489-1507, 1509-1515, 1535-1540, 1571-1591, 1619-1626, 1635-1641, 1647-1655, 1695-1701, 1726-1748, 1750-1757, 1767-1783, 1802-1807, 1809-1822, 1844-1875, 1883-1889, 1922-1929, 1931-1936, 1951-1967, 1978-1989, 1999-2008, 2023-2042, 2056-2083, 2101-2136, 2161-2177 ORF1402 3,4-dihydroxy-2- 18-23, 32-37, 54-63, 65-74, 83-92, E: 3 aa 121-137 E: GSBZB68(121-137): 7/41 198, 217 butanone-4- 107-114, 123-139, 144-155, 157-164, phosphate 191-198, 232-240, 247-272, synthase 284-290, 295-301, 303-309, 311-321, 328-341, 367-376 ORF1473 hemolysin II 4-36, 39-47, 57-65, 75-82, 108-114, F: 1 aa 245-256 F: SALAP76(245-256): 6/41 199, 218 (LukD- 119-126, 135-143, 189-195, 234-244, Leuktoxin) 250-257, 266-272, 311-316 ORF1523 Iron uptake 13-27, 29-44, 46-66, 68-81, 97-116, D: 3 aa 120-135 D: n.d. 309, 337 regulator 138-145 ORF1707 inner membrane 4-23, 57-77, 89-103, 119-125, 132-172, F: 1 aa 104-118 F: SALBC82(104-118): 7/41 200, 219 protein, 60 kDa 179-197, 210-254, 256-265, 281-287 ORF1754 amiB 5-10, 16-24, 62-69, 77-96, 100-115, D: 3 aa 293-312 D: n.d. 310, 338 117-126, 137-156, 165-183, 202-211, 215-225, 229-241, 250-260, 267-273, 290-300, 302-308, 320-333, 336-342, 348-356, 375-382, 384-389 ORF1783 Mrp protein 5-29, 46-52, 70-76, 81-87, 155-170, F: 2 aa 850-860 F: SALAQ36(850-860): 8/41 201, 220 (fmtB) 192-197, 206-213, 215-220, 225-231, 249-258, 273-279, 281-287, 300-306, 313-319, 323-332, 335-341, 344-351, 360-382, 407-431, 443-448, 459-468, 475-496, 513-520, 522-537, 543-550, 556-565, 567-573, 580-585, 593-615, 619-631, 633-642, 670-686, 688-698, 759-766, 768-782, 799-808, 842-848, 868-877, 879-917, 945-950, 979-988, 996-1002, 1025-1036, 1065-1084, 1101-1107, 1113-1119, 1125-1142, 1163-1169, 1183-1189, 1213-1219, 1289-1301, 1307-1315, 1331-1342, 1369-1378, 1385-1391, 1410-1419, 1421-1427, 1433-1447, 1468-1475, 1487-1494, 1518-1529, 1564-1570, 1592-1609, 1675-1681, 1686-1693, 1714-1725, 1740-1747, 1767-1774, 1793-1807, 1824-1841, 1920-1937, 1953-1958, 1972-1978, 1980-1986, 1997-2011, 2048-2066, 2161-2166, 2219-2224, 2252-2257, 2292-2298, 2375-2380, 2394-2399, 2435-2440, 2449-2468 ORF1848 Map-ND2C 4-27, 42-66, 70-76, 102-107, 113-118, E: 5 aa 75-90 E: GSBZB15(75-90): 6/41 202, 221 protein 133-138 ORF1891 ribosomal protein 31-39, 48-54, 61-67, 75-83, 90-98, F: 4 aa 239-257 F: SALAV36(239-257): 19/41 203, 222 L2 (rplB) 103-119, 123-145, 160-167, 169-176, 182-193, 195-206, 267-273 ORF2011 Putative drug 5-27, 79-85, 105-110, 138-165, 183-202, D: 5 aa 205-224 D: n.d. 311, 339 transporter 204-225, 233-259, 272-292, 298-320, 327-336, 338-345, 363-376, 383-398, 400-422, 425-470, 489-495, 506-518, 536-544, 549-554, 562-568, 584-598, 603-623 ORF2027 lactase permease, 10-33, 38-71, 73-103, 113-125, 132-147, E: 2 aa 422-436 E: GSBZF58(422-436): 6/41 204, 223 putative 154-163, 170-216, 222-248, 250-269, 271-278, 287-335, 337-355, 360-374, 384-408, 425-442, 453-465, 468-476, 478-501, 508-529 ORF2087 Hemolysin II 8-27, 52-59, 73-80, 90-99, 104-110, D: 3 aa 126-147 D: n.d. 312, 340 (putative) 117-124, 131-140, 189-209, 217-232, 265-279, 287-293, 299-306 ORF2090 preLukS 8-26, 75-82, 118-126, 136-142, 163-177, F: 2 aa 270-284 F: SALAQ77(270-284): 23/41 205, 224 182-189, 205-215, 221-236, 239-248, 268-274 ORF2092 Hemolysin II 5-22, 30-47, 58-65, 75-81, 87-92, F: 3 aa 238-253 F: SALAQ67(237-252): 10/41 206, 225 (preLUK-F) 99-105, 107-113, 119-126, 189-195, 217-223, 234-244, 250-257, 266-272 ORF2107 Multidrug 10-28, 30-43, 50-75, 80-113, 116-125, D: 9 aa 54-104 D: n.d. 313, 341 resistance protein 136-167, 170-191, 197-245, (putative) 253-329, 345-367, 375-396 ORF2192 Transcriptional 20-31, 46-52, 55-69, 74-79, 89-97, D: 3 aa 15-35 D: n.d. 314, 342 regulator GntR 108-113, 120-128, 141-171, 188-214 family, putative ORF2305 Amino acid 25-79, 91-103, 105-127, 132-149, D: 53 aa 363-393 D: n.d. 315, 343 permease 158-175, 185-221, 231-249, 267-293, 307-329, 336-343, 346-359, 362-405, 415-442, 446-468 ORF2324 Citrate 10-77, 85-96, 99-109, 111-138, 144-155, D: 7 aa 37-83 D: n.d. 316, 344 dransporter 167-176, 178-205, 225-238, 241-247, 258-280, 282-294, 304-309, 313-327, 333-383, 386-402, 405-422, 429-453 ORF2422 Anion transporter 7-26, 28-34, 36-53, 55-73, 75-81, D: 16 aa 275-295 D: n.d. 317, 345 family protein 87-100, 108-117, 121-138, 150-160, 175-181, 184-195, 202-215, 221-247, 265-271, 274-314, 324-337, 341-412, 414-423, 425-440, 447-462, 464-469 ORF2553 SirA 5-22, 54-78, 97-103, 113-123, 130-148, D: 3 aa 1-22 D: n.d. 318, 346 166-171, 173-180, 192-201, 254-261, 266-272, 310-322 ORF2555 omithine cyclode- 20-35, 37-50, 96-102, 109-120, 123-137, E: 2 aa 32-48 E: GSBZB37(32-48): 11/41 207, 226 aminase 141-150, 165-182, 206-224, 237-256, 267-273, 277-291, 300-305, 313-324 ORF2558 Multidrug 11-63, 79-129, 136-191, 209-231, D: 8 aa 84-100 D: n.d. 319, 347 resistance 237-250, 254-276, 282-306, 311-345, efflux proten, 352-373, 376-397 putative ORF2610 Cap5M 4-30, 34-40, 79-85, 89-98, 104-118, D: 13 aa 114-141 D: n.d. 320, 348 124-139, 148-160, 167-178 ORF2613 Cap5P (UDP-N- 4-9, 17-24, 32-38, 44-54, 68-82, B: 3, F: 11 aa 321-341 F: SALAU27(325-337): 9/41 208, 227 acetyl- 89-95, 101-120, 124-131, 136-142, glucosamine 145-157, 174-181, 184-191, 196-204, 2-epimerase) 215-224, 228-236, 243-250, 259-266, 274-281, 293-301, 314-319, 325-331, 355-367, 373-378 ORF2628 Hypothetical 9-15, 28-36, 44-62, 69-88, 98-104, F: 6 aa 694-708 F: SALBD82(1288-1303): 9/41 209, 228 protein 111-136, 139-149, 177-186, 195-217, aa 790-800 224-236, 241-257, 260-278, aa 1288- 283-290, 292-373, 395-408, 411-443, 1305 465-472, 475-496, 503-520, 552-559, 569-589, 593-599, 607-613, 615-636, 648-654, 659-687, 689-696, 721-733, 738-759, 783-789, 795-801, 811-823, 827-836, 839-851, 867-875, 877-883, 890-898, 900-908, 912-931, 937-951, 961-992, 994-1002, 1005-1011, 1016-1060, 1062-1074, 1088-1096, 1101-1123, 1137-1153, 1169-1192, 1210-1220, 1228-1239, 1242-1251, 1268-1275, 1299-1311, 1322-1330, 1338-1361, 1378-1384, 1393-1412, 1419-1425, 1439-1459, 1469-1482, 1489-1495, 1502-1519, 1527-1544, 1548-1555, 1600-1607, 1609-1617, 1624-1657, 1667-1691, 1705-1723, 1727-1742, 1749-1770, 1773-1787, 1804-1813, 1829-1837, 1846-1852, 1854-1864, 1869-1879, 1881-1896, 1900-1909, 1922-1927, 1929-1935, 1942-1962, 1972-2005, 2009-2029, 2031-2038, 2055-2076, 2101-2114, 2117-2124, 2147-2178, 2188-2202, 2209-2217, 2224-2230, 2255-2266, 2271-2280, 2282-2302, 2307-2316, 2319-2324, 2379-2387 ORF2644 PTS system, 8-15, 24-30, 49-68, 80-93, 102-107, F: 4 aa 106-159 F: SALAW60(106-125): 3/41 210, 229 sucrose- 126-147, 149-168, 170-180, 185-193, specific 241-305, 307-339, 346-355, IIBC component 358-372, 382-390, 392-415, 418-425, 427-433, 435-444, 450-472 ORF2654 Oligopeptide 5-61, 72-84, 87-99, 104-109, 124-145, D: 5 aa 182-209 D: n.d. 321, 349 ABC transporter, 158-170, 180-188, 190-216, putative 223-264, 270-275, 296-336, 355-372 ORF2662 maltose ABC 4-21, 71-79, 99-105, 110-121, 143-161, F: 1 aa 306-323 F: SALBC05(306-323): 2/41 211, 230 transporter, 199-205, 219-235, 244-258, putative 265-270, 285-291, 300-308, 310-318, 322-328, 346-351, 355-361, 409-416 ORF2710 sorbitol 4-12, 19-40, 61-111, 117-138, 140-153, B: 2, F: 4 aa 244-257 F: SALAX93(249-256): 6/41 212, 231 dehydrogenase 161-180, 182-207, 226-235, 237-249, 253-264, 267-274, 277-292, 311-323 ORF2742 Hypothetical 4-41, 49-56, 61-67, 75-82, 88-104, D: 188, aa 303-323 D: n.d. 322, 350 protein 114-125, 129-145, 151-165, 171-178, H: 4 187-221, 224-230, 238-250, 252-275, 277-304, 306-385 ORF2780 brnQ 4-29, 41-63, 74-95, 97-103, 107-189, D: 3 aa 26-40 D: n.d. 323, 351 193-209, 220-248, 260-270, 273-299, 301-326, 328-355, 366-397, 399-428 ORF2806 Phage related 10-17, 23-29, 31-37, 54-59, 74-81, F: 3 aa 104-116 F: SALBC34: 1/1 213, 232 protein 102-115, 127-137, 145-152, 158-165, 178-186, 188-196, 203-210, 221-227, 232-237 ORF2900 Conserved 4-27, 34-43, 62-73, 81-90, 103-116, D: 24 aa 360-376 D: n.d. 324, 352 hypothetical 125-136, 180-205, 213-218, 227-235, protein 238-243, 251-259, 261-269, 275-280, 284-294, 297-308, 312-342, 355-380, 394-408, 433-458, 470-510, 514-536, 542-567 ORF2931 conserved 4-19, 43-54, 56-62, 84-90, 96-102, E: 6 aa 22-37 E: GSBZA13(22-37): 7/41 214, 233 hypothetical 127-135, 157-164, 181-187 protein ORF2958 Exotoxin 2 7-19, 26-39, 44-53, 58-69, 82-88, F: 1 aa 154-168 F: SALBB59(154-168): 4/41 215, 234 91-107, 129-141, 149-155, 165-178, 188-194 ORF2970 Surface protein, 9-23, 38-43, 55-60, 69-78, 93-101, H: 5 aa 1-70 H: GSBYU66: n.d. 399, 400 putative 103-112, 132-148, 187-193, 201-208, 216-229, 300-312, 327-352, 364-369, 374-383, 390-396, 402-410, 419-426, 463-475, 482-491 Bacterial surface display: A, LSA250/1 library in fhuA with patient sera 1 (655); B, LSA50/6 library in lamB with patient sera 1 (484); C, LSA250/1 library in fhuA with IC sera 1 (571); E, LSA50/6 library in lamB with IC sera 2 (454); F, LSA50/6 library in lamB with patient sera P1 (1105); G, LSA50/6 library in lamb with IC sera 1 (471); H, LSA250/1 library in fhuA with patient sera 1 (IgA, 708). Ribosome display: D, LSA250/1 library with IC sera (1686). **prediction of antigenic sequences longer than 5 amino acids was performed with the programme ANTIGENIC (Kolaskar and Tongaonkar, 1990). ORF, open reading frame; CRF, reading frame on complementary strand; ARF, alternative reading frame.

TABLE 2c Immunogenic proteins identified by bacterial surface and ribosome display: S. epidermidis. No. of selected Location of S. clones identified Seq ID epidermidis per ORF Immuno- no: antigenic Putative function and genic Serum reactivity with relevant (DNA + protein (by homology) predicted immunogenic aa** screen region region (positive/total) Prot) ARF0172 cation-transporting 4-34, 37-43 D: 6 aa 3-32 D: nd 497, 548 ATPase, E1-E2 family ARF0183 condensing enzyme, 4-22, 24-49 D: 4 aa 1-52 D: nd 498, 549 putative, FabH-related ARF2455 NADH 4-29 D: 3 aa 1-22 D: nd 499, 550 dehydrogenase, putative CRF0001 Unknown 4-14, 16-26 D: 3 aa 5-21 D: nd 500, 551 CRF0002 Unknown 4-13, 15-23, 36-62 D: 5 aa 21-70 D: nd 501, 552 CRF0003 Unknown 4-12, 14-28 D: 3 aa 4-31 D: nd 502, 553 CRF0004 Unknown 5-15, 35-71, 86-94 D: 4 aa 31-72 D: nd 503, 554 CRF0005 Unknown 8-26, 28-34 D: 3 aa: 9-33 D: nd 504, 555 CRF0006 Unknown 4-11, 15-28 D: 3 aa 10-22 D: nd 505, 556 CRF0007 Unknown 4-19, 30-36 D: 3 aa 7-44 D: nd 506, 557 CRF0008 Unknown 10-48 D: 4 aa: 9-44 D: nd 507, 558 CRF0009 Unknown 41883 D: 3 aa 5-14 D: nd 508, 559 CRF0192 Putative protein 4-23, 25-68 C: 4 aa 15-34 C: GSBBM10(15-34): n.d. 445, 446 CRF0275 Putative protein 4-40, 49-65 B: 5 aa 35-68 B: SELAK28(35-68): n.d. 447, 448 CRF0622 Putative protein 4-12, 17-57, 62-70, 75-84, 86-100 C: 4 aa 75-99 C: GSBBR74(76-99): n.d. 449, 450 CRF0879 Putative protein 4-14, 38-44 A: 3, aa 9-40 B: SELAC39(10-40): n.d. 451, 452 B: 10 CRF1004 Putative protein 4-40 A: 3, B: 5 aa 29-65 B: SELAI63(35-63): n.d. 453, 454 CRF2248 Putative protein 4-10, 19-40, 53-64, 74-91 C: 30 aa 74-111 C: GSBBN64(16-35): n.d. 455, 456 CRF2307 Putative protein 4-19, 35-41, 80-89 A: 19 aa 41-87 A: SEFAL47(41-87): n.d. 457, 458 CRF2309 Putative protein 15-21 B: 6 aa 4-16 B: SELAL02(4-16): n.d. 459, 460 CRF2409 Putative protein 6-25 B: 6 aa 2-24 B: SELAB48(5-24): n.d. 461, 462 ORF0005 hypothetical protein 13-27, 33-67, 73-99, 114-129, 132-158, D: 3 aa 105-128 D: nd 509, 560 167-190, 193-234, 237-267, 269-299, 316-330, 339-351, 359-382, 384-423 ORF0008 Streptococcal 9-14, 16-24, 26-32, 41-50, 71-79, B: 2 aa 895-926 B: SELAF79(895-926): 7/12 239, 268 hemagglutinin 90-96, 177-184, 232-237, 271-278, 293-301, 322-330, 332-339, 349-354, 375-386, 390-396, 403-409, 453-459, 466-472, 478-486, 504-509, 518-525, 530-541, 546-552, 573-586, 595-600, 603-622, 643-660, 668-673, 675-681, 691-697, 699-711, 713-726, 732-749, 753-759, 798-807, 814-826, 831-841, 846-852, 871-878, 897-904, 921-930, 997-1003, 1026-1031, 1033-1039, 1050-1057, 1069-1075, 1097-1103, 1105-1111, 1134-1139, 1141-1147, 1168-1175, 1177-1183, 1205-1211, 1213-1219, 1231-1237, 1241-1247, 1267-1273, 1304-1309, 1311-1317, 1329-1335, 1339-1345, 1347-1353, 1382-1389, 1401-1407, 1411-1417, 1447-1453, 1455-1461, 1483-1489, 1491-1497, 1527-1533, 1545-1551, 1556-1561, 1581-1587, 1591-1597, 1627-1638, 1661-1667, 1684-1689, 1691-1697, 1708-1715, 1719-1725, 1765-1771, 1813-1820, 1823-1830, 1835-1856 ORF0038 extracellular 6-25, 29-35, 39-45, 64-71, 82-88, C: 6 aa 136-165 C: GSBBN08(136-165): 1/1 353, 359 elastase precursor 96-102, 107-113, 119-131, 170-176, 186-192, 196-202, 215-220, 243-248, 302-312, 345-360, 362-371, 378-384, 458-470, 478-489, 495-504 ORF0099 hypothetical 6-18, 31-37, 42-49, 51-67, 73-85, D: 5 aa 218-265 D: nd 510, 561 protein 87-93, 102-109, 119-126, 150-157, 170-179, 185-191, 204-214, 217-223, 237-248, 269-275, 278-316, 320-340, 359-365 ORF0101 hypothetical 4-10, 15-27, 67-94, 123-129, 167-173, D: 18 aa 26-109 D: nd 511, 562 protein 179-184, 187-198, 217-222, 229-235, 238-246 ORF0121 C4-dicarboxylate 4-20, 24-62, 73-86, 89-106, 110-122, D: 5 aa 323-379 D: nd 512, 563 transporter, 131-164, 169-193, 204-213, anaerobic, 219-236, 252-259, 263-281, 296-306, putative 318-324, 328-352, 356-397, 410-429 ORF0143 amino acid 25-79, 91-103, 105-127, 132-150, D: 35 aa 247-339 D: nd 513, 564 permease 157-174, 184-206, 208-219, 231-249, 267-294, 310-329, 336-343, 346-405, 417-468 ORF0162 Immunodominant 4-27, 35-45, 52-68, 83-89, 113-119, A: 11, aa 90-227 B: SELAA19(100-118): 1/1 240, 269 Antigen A 133-150, 158-166, 171-176, 198-204, B: 11; B: SELAE24(170-190): 11/12 219-230 C: 153 ORF0201 capa protein, 10-17, 27-53, 81-86, 98-105, 126-135, D: 9 aa 11-53 D: nd 514, 565 putative 170-176, 182-188, 203-217, 223-232, 246-252, 254-269, 274-280, 308-314 ORF0207 Ribokinase (rbsK) 5-11, 15-23, 47-55, 82-90, 98-103, B: 10 aa 20-45 B: SELAQ30 (20-45): 12/12 241, 270 108-114, 126-132, 134-156, 161-186, 191-197, 210-224, 228-235, 239-248, 258-264, 275-290 0RF0288 LrgB 7-28, 34-56, 68-119, 127-146, 149-180, D: 4 aa 112-149 D: nd 515, 566 182-189, 193-200, 211-230 ORF0304 Herpesvirus 8-16, 30-36, 83-106, 116-122, 135-143, D: 8 aa 69-117 D: nd 516, 567 saimiri ORF73 152-165, 177-188, 216-225 homolog, putative ORF0340 nitrate transporter 7-21, 24-93, 101-124, 126-139, D: 5 aa 238-309 D: nd 517, 595 141-156, 163-179, 187-199, 202-242, 244-261, 267-308, 313-322, 340-353, 355-376 ORF0346 hypothetical protein 8-27, 65-73, 87-93, 95-105 D: 8 aa 1-29 D: nd 518, 568 ORF0355 conserved 5-30, 37-43, 57-66, 85-94, 103-111, C: 5 aa 63-86 C: GSBBL39(63-86): 1/1 354, 360 hypothetical 118-125 protein ORF0356 conserved 4-14, 21-53, 60-146, 161-173, 175-182, D: 5 aa 51-91 D: nd 519, 569 hypothetical protein 190-198, 200-211 ORF0406 hypothetical protein 12-32, 35-63, 68-102, 106-137, D: 19 aa 1-48, D: nd 520, 570 139-145, 154-168, 173-185, 203-222, aa 69-102 230-259, 357-364, 366-374 ORF0425 amino acid 40-58, 75-86, 93-110, 117-144, D: 3 aa 401-440 D: nd 521, 571 permease 150-173, 199-219, 229-260, 264-300, 317-323, 329-356, 360-374, 377-390, 392-398, 408-424, 427-452 ORF0442 SceB precursor 7-22, 42-48, 55-66, 83-90, 109-118, C: 38 aa 60-102 C: GSBBM60(65-84): 1/1 355, 361 136-141 ORF0448 SsaA precursor 6-25, 39-47, 120-125, 127-135, C: 170 aa 15-208 C: GSBBN58(81-105): 1/1 356, 362 140-148, 157-168, 200-208, 210-220, C: GSBBL13(167-184): 1/1 236-243, 245-254 C: GSBBL25(22-45): 1/1 ORF0503 Ribosomal protein 31-39, 48-54, 61-67, 75-83, 90-98, A: 1, B: 3 aa 212-273 B: SELAA47(238-259): 12/12 242, 271 L2 103-115, 123-145, 160-167, 169-176, 182-193, 195-206, 267-273 ORF0551 Conserved 5-25, 29-36, 45-53, 62-67, 73-82, A: 16, aa 162-213 B: SELAL12(164-197): 8/12 243, 272 hypothetical protein 84-91, 99-105, 121-142, 161-177, B: 9 187-193, 203-224, 242-251, 266-271, 278-285 ORF0556 hypothetical protein 4-24, 30-41, 43-68, 82-90, 107-114, D: 3 aa 1-26 D: nd 522, 596 123-143, 155-168 ORF0623 Fumble, putative 10-17, 32-38, 55-72, 77-84, 88-96, A: 10, aa 95-150 B: SELAB86(95-128): 3/12 244, 273 126-134, 152-160, 176-185, 190-203, B: 12; 208-214, 217-225, 233-252, C: 1 257-262 ORF0740 Hypothetical protein 18-24, 47-61, 69-83, 90-96, 125-132, B: 3 aa 1093- B: SELAB23(1097-1114): 7/12 245, 274 140-163, 171-188, 222-249, 1114 281-296, 305-315, 322-330, 335-351, 354-368, 390-397, 411-422, 424-431, 451-469, 479-485, 501-507, 517-524, 539-550, 560-568, 588-599, 619-627, 662-673, 678-689, 735-742, 744-749, 780-786, 797-814, 821-827, 839-847, 857-863, 866-876, 902-911, 919-924, 967-982, 1005-1015, 1020-1026, 1062-1070, 1078-1090, 1125-1131, 1145-1150, 1164-1182, 1208-1213, 1215-1234, 1239-1251, 1256-1270, 1298-1303, 1316-1325, 1339-1349, 1362-1369, 1373-1384, 1418-1427, 1440-1448, 1468-1475, 1523-1532, 1536-1542, 1566-1573, 1575-1593, 1603-1619, 1626-1636, 1657-1667, 1679-1687, 1692-1703, 1711-1718, 1740-1746, 1749-1757, 1760-1769, 1815-1849, 1884-1890, 1905-1914, 1919-1925, 1937-1947, 1955-1963, 1970-1978, 2003-2032, 2075-2089, 2117-2124, 2133-2140, 2146-2151, 2161-2167, 2173-2179, 2184-2196, 2204-2220, 2244-2254, 2259-2264, 2285-2296, 2300-2318, 2328-2334, 2347-2354, 2381-2388, 2396-2408, 2419-2446, 2481-2486, 2493-2500, 2506-2516, 2533-2540, 2555-2567, 2576-2592, 2599-2606, 2615-2639, 2647-2655 ORF0757 hypothetical 13-20, 22-28, 33-40, 60-76, 79-86, C: 6 aa 260-284 C: GSBBN01(260-284): 1/1 357, 363 protein 90-102, 112-122, 129-147, 157-170, 178-185, 188-193, 200-205, 218-228, 234-240, 243-250, 265-273, 285-291, 310-316, 330-348, 361-380, 399-405, 427-446, 453-464 ORF0912 DNA mismatch 9-16, 28-39, 47-56, 69-76, 104-121, A: 25 aa 242-304 SEFAT31(242-290): n.d. 441, 442 repair protein 124-130, 137-144, 185-195, 199-214, 238-243, 293-307, 317-337, 351-370, 385-390, 411-428, 472-488, 498-516, 518-525, 528-535, 538-545, 553-559, 563-568, 579-588, 592-607, 615-622, 632-638, 641-648, 658-674, 676-705, 709-720, 727-739, 742-750, 753-760, 768-773, 783-788, 811-819, 827-838 ORF0923 GTP-binding 4-10, 18-27, 42-55, 64-72, 77-92, B: 13 aa 144-163 B: SELAD55(151-163): 8/12 246, 275 protein 114-126, 132-157, 186-196, 206-217, 236-243, 257-280, 287-300, 306-312, 321-328, 338-351, 360-367, 371-382, 385-399 ORF0979 Conserved 4-28, 44-51, 53-84, 88-107, 113-192 A: 9, aa 12-51 B: SELAH01(26-49): 5/12 247, 276 hypothetical protein B: 18 ORF0982 sodium/alanine 13-21, 24-50, 73-84, 91-118, 126-133, D: 3 aa 277-305 D: nd 523, 572 symporter(alsT) 142-149, 156-175, 189-249, 251-273, 294-332, 339-347, 358-381, 393-413, 425-448, 458-463 ORF1230 Signal peptidase I 6-33, 44-59, 61-69, 74-82, 92-98, D: 14 aa 1-53 D: nd 524, 573 133-146, 163-175 ORF1232 Exonuclease 4-12, 16-32, 36-48, 50-65, 97-127, B: 6 aa 188-219 B: SELAA13(188-216): n.d. 443, 444 RexA 136-142, 144-165, 176-190, 196-202, 211-222, 231-238, 245-251, 268-274, 280-286, 305-316, 334-356, 368-376, 395-402, 410-417, 426-440, 443-449, 474-486, 499-508, 510-525, 540-549, 568-576, 608-617, 624-639, 646-661, 672-678, 688-703, 706-717, 727-734, 743-755, 767-773, 783-797, 806-814, 830-839, 853-859, 863-871, 877-895, 899-918, 935-948, 976-990, 998-1007, 1020-1030, 1050-1062, 1070-1077, 1111-1125, 1137-1149, 1153-1160, 1195-1211 ORF1284 permease PerM, 10-60, 72-96, 103-109, 127-133, D: 27 aa 55-106 D: nd 525, 574 putative 146-177, 182-189, 196-271, 277-289, 301-319, 323-344, 347-354 ORF1319 2-oxoglutarate 9-31, 36-45, 59-67, 71-81, 86-94, B: 5; C: 1 aa 400-413 B: SELAF54(404-413): 11/12 248, 277 decarboxylase 96-107, 111-122, 127-140, 153-168, (menD) 180-211, 218-224, 226-251, 256-270, 272-289, 299-305, 310-323, 334-341, 345-353, 358-364, 369-379, 384-390, 396-410, 417-423, 429-442, 454-464, 470-477, 497-505, 540-554 ORF1326 autolysin AtlE 6-25, 40-46, 75-81, 150-155, 200-205, B: 7; C: 5 aa 1282- B: SELAD20(1282-1298): 249, 278 (lytD) 237-243, 288-295, 297-306, 1298 10/12 308-320, 341-347, 356-363, 384-391, 417-429, 440-452, 465-473, 481-514, 540-546, 554-560, 565-577, 585-590, 602-609, 611-617, 625-634, 636-643, 661-668, 676-684, 718-724, 734-742, 747-754, 766-773, 775-781, 785-798, 800-807, 825-832, 840-857, 859-879, 886-892, 917-923, 950-956, 972-978, 987-1002, 1028-1035, 1049-1065, 1071-1099, 1111-1124, 1150-1172, 1185-1190, 1196-1207, 1234-1241, 1261-1271, 1276-1281, 1311-1320, 1325-1332 ORF1333 quinol oxidase 4-27, 33-55, 66-88 D: 4 aa 3-93 D: nd 526, 575 polypeptide iv (ec 1.9.3.—) (quinol oxidase aa 3-600, subunit qoxd) ORF1356 hypothetical protein 9-36, 44-67, 74-97, 99-149, 161-181, D: 32 aa 54-95 D: nd 527, 597 189-198, 211-224, 245-253, 267-273, 285-290, 303-324, 342-394, 396-427 ORF1373 dihydrolipoamide 33-39, 42-78, 103-109, 126-136, A: 3, B: 1 aa 124-188 A: SEFAP57(124-188): 2/12 250, 279 acetyltransferase 184-191, 225-232, 258-279, 287-294, 306-315, 329-334, 362-379, 381-404, 425-430 ORF1381 hypothetical protein 21-45, 62-67, 74-106, 108-142, D: 5 aa 7-44 D: nd 528, 576 154-160, 230-236, 245-251, 298-305 ORF1420 Muts2 protein, 8-32, 34-41, 46-55, 70-76, 81-89, B: 7 aa 581-608 B: SELAM40(581-604): 9/12 251, 280 putative 97-115, 140-148, 153-159, 165-171, 175-188, 207-239, 256-276, 280-289, 297-319, 321-335, 341-347, 352-360, 364-371, 384-411, 420-440, 449-460, 495-502, 505-516, 560-566, 573-588, 598-605, 607-614, 616-624, 674-694, 702-717 ORF1443 cell division 61-66, 111-117, 148-155, 173-182, D: 4 aa 175-229 D: nd 529, 577 protein (divIB) 194-224, 263-293, 297-303, 313-321, 334-343, 345-356, 375-381, 384-395, 408-429, 448-454 ORF1500 Cell division protein 100-107, 154-167, 182-193, 200-206, A: 2, B: 3 aa 77-182 B: SELAP37(139-162): 9/12 252, 281 FtsY 223-231, 233-243, 249-257, 265-273, 298-310, 326-336, 343-362, 370-384 ORF1665 amino acid ABC 4-25, 44-55, 66-76, 82-90, 93-99, D: 5 aa 1-52 D: nd 530, 578 transporter, 104-109, 176-209, 227-242, 276-283, permease protein 287-328, 331-345, 347-376, 400-407, 409-416, 418-438, 441-474 ORF1707 putative host cell 12-31, 40-69, 129-137, 140-151, D: 4 aa 20-76 D: nd 531, 598 surface-exposed 163-171, 195-202, 213-218 lipoprotein ORF1786 D-3- 4-10, 16-32, 45-55, 66-78, 87-95, D: 5 aa 400-442 D: nd 532, 579 phosphoglycerate 103-115, 118-124, 135-150, 154-161, dehydrogenase, 166-174, 182-193, 197-207, putative 225-231, 252-261, 266-304, 310-315, 339-347, 351-359, 387-402, 411-423, 429-436, 439-450, 454-464, 498-505, 508-515 ORF1849 yhjN protein 8-51, 53-69, 73-79, 85-132, 139-146, D: 5 aa 254-301 D: nd 533, 580 148-167, 179-205, 212-224, 231-257, 264-293, 298-304, 309-317, 322-351 ORF1877 protein-export 6-19, 26-39, 41-51, 59-67, 72-85, D: 7 aa 367-409 D: nd 534, 581 membrane protein 91-98, 104-111, 120-126, 147-153, SecD (secD-1) 158-164, 171-178, 199-209, 211-218, 233-249, 251-257, 269-329, 362-368, 370-385, 392-420, 424-432, 454-489, 506-523, 534-539, 550-556, 563-573, 576-596, 603-642, 644-651, 655-666, 685-704, 706-733, 747-753 ORF1912 unknown conserved 23-35, 37-70, 75-84, 90-112, 129-135, D: 4 aa 131-187 D: nd 535, 582 protein 137-151, 155-180, 183-209, (conserved) 211-217, 219-225, 230-248, 250-269, 274-284, 289-320, 325-353, 357-371, 374-380, 384-399, 401-411, ORF2015 Trehalose-6- 8-17, 30-54, 82-89, 94-103, 157-166, A: 3, B: 8 aa 465-498 B: SELAH62(465-498): 5/12 253, 282 phosphate 178-183, 196-204, 212-219, hydrolase 222-227, 282-289, 297-307, 345-364, 380-393, 399-405, 434-439, 443-449, 453-475, 486-492, 498-507, 512-535, 538-548 ORF2018 Glucose-6- 4-16, 21-27, 39-51, 60-69, 76-83, B: 17 aa 250-287 B: SELAI19(250-279): 3/12 254, 283 phosphate 1-DH 97-118, 126-132, 159-167, 171-177, 192-204, 226-240, 247-259, 281-286, 294-305, 314-320, 330-338, 353-361, 367-372, 382-392, 401-413, 427-434, 441-447, 457-463 ORF2040 LysM domain 51-56, 98-108, 128-135, 138-144, D: 23 aa 259-331 D: nd 536, 583 protein protein 152-158, 177-192, 217-222, 232-251, 283-305, 406-431, 433-439 ORF2098 PilB related 13-18, 36-43, 45-50, 73-79, 95-100, A: 60 aa 1-57 A: SEFAQ50(15-57): 5/12 255, 284 protein 111-126, 133-139 ORF2139 sodium: sulfate 7-12, 22-97, 105-112, 121-128, D: 41 aa 42-118 D: nd 537, 584 symporter family 130-146, 152-164, 169-189, 192-203, protein, putative 211-230, 238-246, 260-281, 304-309, 313-325, 327-357, 367-386, 398-444, 447-476, 491-512 ORF2172 SceB precursor 4-23, 28-34, 38-43, 45-51, 63-71, A: 438, aa 6-215 B: SELAH53(188-209): 3/12 256, 285 (lytE) 85-96, 98-112, 118-126, 167-174, B: 40, 179-185, 219-228, 234-239, 256-263 D: 4 ORF2200 zinc ABC 4-31, 33-40, 48-64, 66-82, 92-114, D: 19 aa 162-225 D: nd 538, 585 transporter, 118-133, 137-159, 173-246, 248-266 permease protein, putative ORF2248 membrane protein, 4-11, 17-34, 72-78, 127-137, 178-227, D: 17 aa 1-59, D: nd 539, 586 MmpL family, 229-255, 262-334, 352-380, aa 159-225, putative 397-405, 413-419, 447-454, 462-467, aa 634-674 478-490, 503-509, 517-558, 560-568, 571-576, 582-609, 623-629, 631-654, 659-710, 741-746, 762-767, 771-777, 788-793, 856-867 ORF2260 Unknown conserved 5-10, 18-29, 31-37, 66-178, 196-204, B: 4 aa 123-142 B: SELAG77(123-142): 12/12 257, 286 protein in 206-213 others ORF2282 conserved 16-22, 41-50, 52-64, 66-74, 89-95, A: 4 aa 51-97 A: SEFAR88(51-97): 3/12 258, 287 hypothetical protein 107-114, 123-130, 135-159, 167-181, 193-199, 223-231, 249-264, 279-289 ORF2376 DivIC homolog, 27-56, 102-107, 111-116 D: 7 aa 15-58 D: nd 540, 587 putative ORF2439 membrane-bound 4-9, 11-26, 36-56, 59-73, 83-100, A: 459, aa 10-217 B: SELAC31(75-129): 12/12 259, 288 lytic murein 116-130, 148-163, 179-193, 264-270, B: 2, transglycosidase 277-287, 311-321 D: 53 D, putative ORF2493 conserved 4-29, 37-77, 80-119 D: 6 aa 69-113 D: nd 541, 588 hypothetical protein ORF2535 ATP-binding 5-28, 71-81, 101-107, 128-135, D: 8 aa 1-65 D: nd 542, 589 cassette 146-52, 178-188, 209-214, 224-233, transporter-like 279-294, 300-306, 318-325, 342-347, protein, putative 351-357 ORF2627 cation- 8-31, 34-80, 125-132, 143-153, D: 3 aa 61-105 D: nd 543, 590 transporting 159-165, 176-189, 193-198, 200-206, ATPase, E1-E2 215-242, 244-262, 264-273, family, putative 281-289, 292-304, 318-325, 327-338, 347-371, 404-416, 422-429, 432-450, 480-488, 503-508, 517-525, 539-544, 551-562, 574-587, 600-631, 645-670 ORF2635 Hypothetical 4-10, 17-24, 26-42, 61-71, 90-96, A: 2, B: 2 aa 139-169 B: SELAB63(138-163): 7/12 260, 289 protein 102-111, 117-125, 158-164, 173-182, 193-201, 241-255, 268-283, 289-298, 305-319, 340-353, 360-376, 384-390, 394-406 ORF2669 Hypothetical 4-21, 35-42, 85-90, 99-105, 120-125, A: 14, aa 22-81 B: SELAE27(22-51): 5/12 261, 290 protein 148-155, 175-185, 190-196, B: 8 205-210, 217-225 ORF2671 Hypothetical protein 4-23, 43-49, 73-84, 93-98, 107-113, A: 44, aa 23-68 B: SELAD21(36-61): 5/12 262, 291 156-163, 179-190, 197-204, 208-218, B: 14 225-231, 248-255 ORF2673 Hypothetical 4-20, 65-71, 99-105, 148-155, 171-182, A: 16, aa 23-68 B: SELAE25(23-54): 2/12 263, 292 protein 190-196, 204-210, 221-228, B: 3 240-246 ORF2694 Hypothetical 4-26, 93-98, 121-132, 156-163, A: 19, aa 25-82 B: SELAB26(27-60): 5/12 264, 293 protein 179-192, 198-204, 212-220, 225-238 B: 30 ORF2695 Hypothetical 4-26, 43-50, 93-98, 107-113, 156-163, A: 7 aa 22-78 A: SEFAH77(22-66): 6/12 265, 294 protein 179-190, 198-204, 212-218, 225-231, 247-254 ORF2719 two-component 5-52, 60-71, 75-84, 91-109, 127-135, B: 4 aa 123-132 B: SELAA62(123-132): 6/12 266, 295 sensor histidine 141-156, 163-177, 185-193, kinase, putative 201-214, 222-243, 256-262, 270-279, 287-293, 298-303, 321-328, 334-384, 390-404, 411-418, 427-435, 438-448, 453-479, 481-498, 503-509 ORF2728 Accumulation- 4-13, 36-44, 76-86, 122-141, 164-172, A: 265, aa 803-1001 B: SELAA10(850-878): 11/12 267, 296 associated protein 204-214, 235-242, 250-269, B: 448; 291-299, 331-337, 362-369, 377-396, C: 4, D: 9 419-427, 459-469, 505-524, 547-555, 587-597, 618-625, 633-652, 675-683, 715-727, 740-753, 761-780, 803-811, 842-853, 962-968, 1006-1020 ORF2740 lipase precursor 4-21, 190-200, 218-228, 233-241, C: 3 aa 110-177 C: GSBBL80(110-177): 1/1 358, 364 243-261, 276-297, 303-312, 316-325, 346-352, 381-387, 436-442, 457-462, 495-505, 518-532, 543-557, 574-593 ORF2764 oligopeptide ABC 14-36, 62-131, 137-147, 149-162, D: 4 aa 6-41 D: nd 544, 591 transporter, 164-174, 181-207, 212-222, 248-268, permease protein, 279-285 putative ORF2767 unknown conserved 7-20, 22-35, 40-50, 52-61, 63-92, D: 4 aa 276-316 D: nd 545, 592 protein in 94-101, 103-126, 129-155, 161-178, others 192-198, 200-208, 210-229, 232-241, 246-273, 279-332, 338-359, 369-383 ORF2809 sodium: sulfate 4-29, 37-53, 56-82, 87-100, 108-117, D: 9 aa 266-317, D: nd 546, 593 symporter family 121-138, 150-160, 175-180, aa 357-401 protein 189-195, 202-214, 220-247, 269-315, 324-337, 341-355, 361-412, 414-423, 425-440, 447-467 ORF2861 putative 7-13, 20-32, 37-90, 93-103, 107-126, D: 11 aa 137-185 D: nd 547, 594 transmembrane 129-155, 159-173, 178-189, efflux rotein 195-221, 234-247, 249-255, 268-303, 308-379 Bacterial surface display: A, LSE150 library in fhuA with patient sera 2 (957); B, LSE70 library in lamB with patient sera 2 (1420); C, LSE70 library in lamB with patient sera 1 (551). Ribosome display: D, LSE150 in pMAL4.31 with P2 (1235). **prediction of antigenic sequences longer than 5 amino acids was performed with the programme ANTIGENIC (Kolaskar and Tongaonkar, 1990). ORF, open reading frame; ARF, alternative reading frame; CRF, reading frame on complementary strand. ORF, open reading frame; CRF, reading frame on complementary strand.

TABLE 2d Immunogenic proteins identified by bacterial surface and ribosome display: S. aureus (new annotation) No. of Location of S. Putative selected Identified Seq aureus Old function clones per immuno- ID no: antigenic ORF (by ORF and genic Serum reactivity with relevant (DNA + protein number homology) predicted immunogenic aa** screen region region (positive/total) Prot) SaA0003 ORF2967 repC 7-19, 46-57, 85-91, 110-117, 125-133, B: 3, C: 14; aa 9-42 C: GSBYI53(9-42): 1/1 394, & 140-149, 156-163, 198-204, F: 29 aa 156-241 C: GSBYG39(156-241): 1/1 396 ORF2963 236-251, 269-275, 283-290, 318-323, aa 300-314 C: GSBYM94(343-420): 26/30 347-363 aa 343-420 ORF0123 ORF1909 unknown 4-10, 25-30, 38-57, 91-108, 110-123, B: 3, E: 7, aa 145-163 B: GSBXF80(150-163): 5/27 409, ~18 aa at 125-144, 146-177, 179-198, G: 1 E: GSBZC17(150-163): 25/41 410 N- 216-224, 226-233 terminus ORF0160 ORF1941 unknown 4-26, 34-70, 72-82, 86-155, 160-166, A: 1 aa 96-172 A: GSBXO07(96-172): 5/30 411, −16 aa at 173-205, 207-228, 230-252, 412 N- 260-268, 280-313 terminus ORF0657 ORF LPXTGVI 9-33, 56-62, 75-84, 99-105, 122-127, A: 2, B: 27, aa 526-544 B: GSBXE07-bdb1(527-542): 413, unknown protein 163-180, 186-192, 206-228, F: 15 11/71 414 233-240, 254-262, 275-283, 289-296, F: SALAX70(526-544): 11/41 322-330, 348-355, 416-424, 426-438, 441-452, 484-491, 541-549, 563-569, 578-584, 624-641 ORF1050 ORF1307 unknown 45-68, 72-79, 91-101, 131-142, A: 1, H: 45 aa 53-124 A: GSBXM26(53-124): 7/30 415, −4 aa at 144-160, 179-201 416 N- terminus ORF1344 ORF0212 NifS 13-26, 40-49, 61-68, 92-112, 114-123, A: 11 aa24-84 A: GSBXK59-bmd21(24-84): 417, −10 aa at protein 138-152, 154-183, 194-200, 6/29 418 N- homolog 207-225, 229-240, 259-265, 271-284, terminus 289-309, 319-324, 330-336, 346-352, 363-372 ORF1632 ORF1163 SdrH 4-31, 50-55, 243-257, 259-268, B: 6, E: 11, aa 101-115 B: GSBXG53(164-182): 39/71 419, −4 aa at homolog 298-316, 326-335, 364-370, 378-407 F: 34 aa 115-139 F: SALAP07(101-115): 11/41 420 N- aa 158-186 terminus ORF2180 ORF0594 LPXTGIV 9-17, 24-45, 67-73, 82-90, 100-107, A: 3, C: 3, aa 491-587 A: GSBXS61(491-555): 1/1 421, −2 aa at protein 117-134, 137-145, 158-168, 176-183, E: 6, F: 2, aa 633-715 A: GSBXL64(494-585): 1/1 422 N- 188-194, 206-213, 223-231, H: 6 aa 702-757^(#) A: GSBXS92(758-841): 1/1 terminus 243-248, 263-270, 275-282, 298-304, aa 758-830 A: bmd4(702-757): 16/30^(#) 344-355, 371-377, 382-388, (aa (A: bmd4(830-885): 16/30)^(#) 427-433, 469-479, 500-505, 534-559, 830-885)^(#) F: SALBC43(519-533): 4/41 597-607, 662-687, 790-815, 918-943, 1032-1037, 1046-1060, 1104-1112, 1128-1137, 1179-1184, 1197-1204, 1209-1214, 1221-1239 ORF2184 ORF0590 FnbpB 10-29, 96-116, 131-137, 146-158, A: 2, C: 4, aa 694-769 A: GSBXM62(694-769): 28/28 423, −8 aa at 167-173, 177-182, 185-191, 195-201, G: 9 aa 774-847 A: GSBXR22(774-847): 1/1 424 N- 227-236, 260-266, 270-284, terminus 291-299, 301-312, 348-356, 367-376, 382-396, 422-432, 442-453, 480-487, 497-503, 519-527, 543-548, 559-565, 579-585, 591-601, 616-623, 643-648, 657-663, 706-718, 746-758, 791-796, 810-817, 819-825, 833-839, 847-853, 868-885, 887-895, 919-932 ORF2470 ORF0299 Conserved 4-27, 36-42, 49-55, 68-73, 94-101, C: 3 aa 400-441 C: GSBYH60(400-441): 28/31 425, −14 aa at hypo- 131-137, 193-200, 230-235, 270-276, 426 N- thetical 294-302, 309-324, 334-344, terminus protein 347-364, 396-405, 431-437, 498-508, 513-519, 526-532, 539-544, 547-561, 587-594, 618-630, 642-653, 687-699, 713-719, 752-766 ORF2498 ORF0267 Conserved 8-19, 21-44, 63-76, 86-92, 281-286, D: 12, F: 6 aa 358-411 D: 17/21 427, ORF app. hypo- 303-322, 327-338, 344-354, 364-373, aa 588-606 F: SALAT38(895-909): 8/41 428 580 aa thetical 379-394, 405-412, 453-460, aa 895-909 longer at protein 501-506, 512-518, 526-542, 560-570, N 577-583, 585-604, 622-630, terminus; 645-673, 677-691, 702-715, 727-741, plus 748-753, 770-785, 789-796, other 851-858, 863-869, 876-881, 898-913, changes 917-924, 979-986, 991-997, 1004-1009, 1026-1041, 1045-1052, 1107-1114, 1119-1125, 1132-1137, 1154-1169, 1173-1192, 1198-1204, 1240-1254, 1267-1274, 1290-1298, 1612-1627 ORF2548 ORF2711 IgG 4-37, 44-53, 65-71, 75-82, 105-112, A: 55, aa 1-123 A: GSBXK68(1-73): 21/30 429, −12 aa at binding 126-132, 136-143, 164-170, 184-190, B: 54, aa 207-273 A: GSBXK41(35-123): 1/1 430 N- protein 194-201, 222-232, 242-248, C: 35, aa 310-410 A: GSBXN38(207-273): 19/30 terminus A 252-259, 280-291, 300-317, 413-420, F: 59, A: GSBXL11(310-363): 10/30 452-460, 485-503 G: 56, B: GSBXB22(394-406): 37/71 H: 38 F: SALAM17(394-406): 29/41 ORF2746 ORF2507 homology 4-9, 12-17, 40-46, 91-103, 106-113, A: 1, H: 13 aa 63-126 A: GSBXO40(66-123): 8/29 431, −3 aa at with 116-125, 150-160, 172-177, 182-188, 432 N- ORF1 195-206, 241-261, 263-270, terminus 277-285, 287-294 ORF2797 ORF2470 unknown 13-32, 40-75, 82-95, 97-112, 115-121, B: 3, E: 2, aa 159-176 B: GSBXE85(159-176): 11/27 433, −24 aa at 124-154, 166-192, 201-225, F: 13, H: 3 aa 325-339 F: SALAQ47(159-176): 8/41 434 N- 227-252, 268-273, 288-297, 308-375, terminus 379-434 ORF2960 ORF2298 putative 8-31, 35-44, 106-113, 129-135, C: 101, aa 1-80 C: GSBYG32(1-80): 6/7 435, −5 aa at Exotoxin 154-159, 168-178, 203-215, 227-236, E: 2, H: 58 aa 48-121 C: GSBYG61-bhe2(48-116): 436 N- 240-249, 257-266, 275-281, aa 98-190 26/30 terminus 290-296, 298-305, 314-319, 327-334 C: GSBYN80(98-190):: 13/17 ORF2963 ORF2295 putative 8-23, 35-41, 64-70, 81-87, 109-115, C: 3, E: 3, aa 17-95 C: GSBYJ58(17-95): 9/15 437, −5 aa at Exotoxin 121-132, 150-167, 177-188, 194-201, G: 1 438 N- 208-216, 227-233, 238-248, terminus 265-271, 279-285 ORF3200 ORF1331 putative 8-32, 45-52, 92-103, 154-159, 162-168, A: 11, aa A: GSBXL07(8543-8601): 6/28 439, +8506 aa extra- 207-214, 232-248, 274-280, B: 11, 8543-8601 440 at N- cellular 297-303, 343-349, 362-375, 425-442, C: 36, aa terminus matrix 477-487, 493-498, 505-512, H: 32 8461-8475 binding 522-533, 543-550, 558-564, 568-574, protein 580-600, 618-630, 647-652, 658-672, 692-705, 711-727, 765-771, 788-798, 812-836, 847-858, 870-898, 903-910, 1005-1015, 1018-1025, 1028-1036, 1058-1069, 1075-1080, 1095-1109, 1111-1117, 1119-1133, 1166-1172, 1183-1194, 1200-1205, 1215-1222, 1248-1254, 1274-1280, 1307-1317, 1334-1340, 1381-1391, 1414-1420, 1429-1439, 1445-1467, 1478-1495, 1499-1505, 1519-1528, 1538-1550, 1557-1562, 1572-1583, 1593-1599, 1654-1662, 1668-1692, 1701-1707, 1718-1724, 1738-1746, 1757-1783, 1786-1793, 1806-1812, 1815-1829, 1838-1848, 1853-1860, 1875-1881, 1887-1893, 1899-1908, 1933-1940, 1952-1961, 1964-1970, 1977-1983, 1990-1996, 2011-2018, 2025-2038, 2086-2101, 2103-2117, 2177-2191, 2195-2213, 2220-2225, 4*2237-2249, 2273-2279, 2298-2305, 2319-2327, 2349-2354, 2375-2381, 2391-2398, 2426-2433, 2436-2444, 2449-2454, 2463-2469, 2493-2499, 2574-2589, 2593-2599, 2605-2611, 2615-2624, 2670-2684, 2687-2698, 2720-2727, 2734-2754, 2762-2774, 2846-2866, 2903-2923, 2950-2956, 2985-2998, 3011-3031, 3057-3064, 2*3102-3117, 3137-3143, 3186-3195, 3211-3219, 3255-3270, 3290-3300, 3327-3334, 3337-3343, 3390-3396, 3412-3419, 3439-3446, 3465-3470, 3492-3500, 3504-3510, 3565-3573, 3642-3650, 3691-3698, 3766-3775, 3777-3788, 3822-3828, 3837-3847, 3859-3864, 3868-3879, 3895-3902, 3943-3951, 3963-3971, 3991-3997, 4018-4030, 4054-4060, 4074-4099, 4123-4129, 4147-4153, 4195-4201, 4250-4255, 4262-4267, 4270-4277, 4303-4310, 4321-4330, 4343-4352, 4396-4408, 4446-4451, 4471-4481, 4503-4509, 4516-4534, 4596-4604, 4638-4658, 4698-4710, 4719-4732, 4776-4783, 4825-4833, 4851-4862, 4882-4888, 4894-4909, 4937-4942, 5047-5054, 5094-5100, 5102-5112, 5120-5125, 5146-5153, 5155-5164, 5203-5214, 5226-5236, 5278-5284, 5315-5321, 5328-5342, 5348-5359, 5410-5420, 5454-5466, 5481-5489, 5522-5538, 5597-5602, 5607-5614, 0″5623-5929, 5650-5665, 5707-5719, 5734-5742, 5772-5778, 5785-5790, 5833-5845, 5857-5863, 5899-5904, 5908-5921, 5959-5971, 5981-5989, 6010-6017, 6034-6043, 6058-6064, 6112-6120, 6154-6169, 6210-6217, 6231-6240, 6261-6268, 6288-6294, 6318-6324, 6340-6349, 6358-6369, 6402-6407, 6433-6438, 6483-6493, 6513-6519, 6527-6546, 6561-6574, 6599-6608, 6610-6616, 6662-6673, 6696-6705, 6729-6743, 6769-6775, 6792-6801, 6819-6828, 6840-6846, 6860-6870, 6915-6928, 6966-6972, 7021-7028, 7032-7047, 7096-7101, 7109-7117, 7138-7149, 7157-7162, 7201-7206, 7238-7253, 7283-7294, 7296-7302, 7344-7365, 7367-7376, 7389-7404, 7413-7433, 7475-7482, 7493-7500, 7535-7549, 7596-7608, 7646-7651, 7661-7678, 7722-7731, 7741-7754, 7764-7769, 7776-7782, 7791-7806, 7825-7837, 7862-7875, 7891-7897, 7922-7931, 7974-7981, 7999-8005, 8039-8045, 8049-8065, 8070-8075, 8099-8112, 8119-8125, 8151-8158, 8169-8181, 8226-8232, 8258-8264, 8291-8299, 8301-8310, 8325-8335, 8375-8389, 8394-8400, 8405-8412, 8421-8436, 8478-8485, 8512-8521, 8528-8538, 8564-8579, 8587-8594, 8603-8615, 8626-8637, 8640-8646, 8657-8672, 8684-8691, 8725-8736, 8748-8761, 8777-8783, 8794-8799, 8810-8825, 8851-8862, 8874-8887, 8903-8912, 8914-8926, 8933-8943, 8954-8960, 8979-8988, 9004-9011, 9035-9041, 9056-9069, 9077-9086, 9088-9096, 9106-9111, 9124-9133, 9183-9191, 9224-9231, 9235-9241, 9250-9265, 9279-9290, 9295-9300, 9326-9343, 9408-9414, 9422-9427, 9435-9441, 9455-9461, 9507-9517, 9532-9538, 9580-9589, 9594-9600, 9614-9623, 9643-9648, 9665-9683, 9688-9700, 9720-9726, 9742-9758, 9767-9775, 9795-9800, 9817-9835, 9842-9847, 9912-9919, 9925-9938, 9943-9963, 9970-10009, 10025-10031, 10037-10043, 10045-10063, 10066-10073, 10117-10124, 10126-10136, 10203-10210, 10218-10225, 10232-10242, 10287-10292, 10303-10323, 10352-10360, 10385-10396,10425-10431, 10452-10459, 10480-10485 Bacterial surface display: A, LSA250/1 library in fhuA with patient sera 1 (655); B, LSA50/6 library in lamB with patient sera 1 (484); C, LSA250/1 library in fhuA with IC sera 1 (571); E, LSA50/6 library in lamB with IC sera 2 (454); F, LSA50/6 library in lamB with patient sera P1 (1105); G, LSA50/6 library in lamb with IC sera 1 (471). Ribosome display: D, LSA250/1 library with IC sera (1686). **prediction of antigenic sequences longer than 5 amino acids was performed with the programme ANTIGENIC (Kolaskar and Tongaonkar, 1990); ^(#)identical sequence present twice in ORF.

TABLE 3 Serological proteome analysis of S. aureus surface proteins using human sera a) S. aureus/agr “stress conditions” Infant pool IC40 IC35, N26, C4 C2, 5, 6, 10, 12 N22 1:10,000 Spot ID/sera 1:20,000 1:50,000 each 1:10,000 IC40 1:50,000 PCK2 + + − + PCK4 + +++ − +++ PCK5 − (+) − + PCK6 + + − + IC35, 40 P-pool Infant pool 1:50,000 (P6, 18, 25, 28, 29) C2, 5, 6, 10, 12 Spot ID/sera N22 1:10,000 1:50,000 each 1:10,000 PAC1 ++ ++ − PAC2 ++ +++ − PAC3 − + − PAC5 − ++ − P-pool IC pool/IgG IC pool/IgA (P6, 18, 25, 28, 29) Infant 14 (N26, IC34, 35) (N26, IC34, 35) Spot ID/sera 1:50,000 each 1:10,000 1:30,000 each 1:30,000 each PAC11 ++ − ++ ++ PAC12 ++ − ++ ++ PAC13 − − − ++ PAC14 − − + + PAC15 − − +++ +++ PAC16 + − + + PAC17 + − + + PAC18 ++ − − − PAC19 − − ++ ++ PAC20 ++ − − − POV31 +++ − − − POV32 + − − − POV33 + − − − POV34 + − − − POV35 + − − − POV36 + − − − POV37 ++ − − − POV38 ++ − − − POV39 +++ − − − POV40 +++ − − − b) S. aureus/COL “standard conditions” IC pool (N26, IC34, 35) IC35 P18 P25 P1 P29 Infant 18 Spot ID/sera 1:30,000 each 1:20,000 1:10,000 1:10,000 1:5,000 1:2,500 1:10,000 POV2 +++ +++ +++ +++ +++ − − POV3.1 +++ +++ +++ +++ +++ − − POV3.2 +++ +++ +++ +++ +++ − − POV4 + +++ − − − − − POV7 − − +++ − − − − POV10 − ++ (+) (+) − (+) − POV12 − − − − − +++ − POV13 ++ +++ +++ +++ ++ ++ − POV14 ++ +++ +++ ++ ++ ++ − POV15 + + − + (+) − − c) S. aureus/COL “stress conditions” P-pool (P6, 18, 25, 28, 29) IC34 + IC35 P18 P29 Infant 14 Spot ID/sera 1:50,000 each 1:20,000 each 1:10,000 1:10,000 1:10,000 POV16 − +++ − − − POV17 − +++ (+) − − POV18 + − ++ − − POV19 (+) − +++ − − POV21 − − + − − POV23 − + − − − POV24 − + − − − POV25 + − − − −

TABLE 4 S. aureus antigens identified by MALDI-TOF-MS sequencing (ORFs in bold were also identified by bacterial surface display) Prediction of antigenic regions in selected antigens identified by serological proteome analysis using human sera S. aureus protein Seq ID no: spot ID (ORF no./abbrev.) Putative function (by homology) (DNA, Prot) Putative localization PCK2 ORF0599 Glycinamide —ribosyl synthase 107, 108 cytoplasmic PCK5 ORF0484 yitU conserved hypoth. protein (yitU) 109, 110 cytoplasmic PCK6 ORF2309 membrane-associated malate-quinone 111, 112 peripheral membrane mqo oxidase POV2 ORF0766 aux1 protein phosphatase contributing to methicilin 113, 114 trans-membrane resistance POV4, 17 ORF0078 EF- C-terminal part of 44 kDa protein similar 115, 116 cytoplasmic/secreted PAC14, 19 Tu to elongation factor Tu POV5¹⁾ ORF0782 3-ketoacyl-acyl carrier protein reductase 117, 118 cytoplasmic (fabG) POV7 ORF0317 SecA protein transport across the membrane 39, 91 cytoplasmic SecA POV10 ORF1252 yrzC hypothetical BACSU 11.9 kd protein 119, 120 cytoplasmic (upf0074 (rff2) family) POV12 ORF0621 pdhB dihydrolipoamide acetyltransferase 121, 122 cytoplasmic (pdhB) POV14 ORF0072 rpoB DNA-directed RNA polymerase β 125, 126 cytoplasmic POV15 ORF0077 EF-G 85 kD vitronectin binding protein 127, 128 cytoplasmic POV18 not found general stress protein YLY1 129, 130 cytoplasmic YLY1 POV30¹⁾ ORF0069 RL7 ribosomal protein L7 131, 132 cytoplasmic POV21 ORF0103 probable hexulose-6-phosphate synthase 133, 134 cytoplasmic yckG (yckG) POV24 ORF0419 conserved hypothetical protein (yurX) 137, 138 cytoplasmic yurX POV25 ORF2441 glucose inhibited division protein a (gidA) 139, 140 cytoplasmic gidA PAC1 ORF1490 protein export protein prsa precursor 173, 174 periplasmic prsA (prsA) PAC2 ORF1931 periplasmic molybdate binding protein 175, 176 surface ModA (ModA) PAC3 ORF2053 heavy metal dependent transcriptional 177, 178 cytoplasmic activator, putative regulator of multidrug resistance efflux pump pmrA PAC5 ORF2233 pyruvate oxidase (ydaP) 179, 180 cytoplasmic ydaP PAC11 ORF1361 LPXTGV, extracellularmatrix-bdg. 3, 56 surface PAC12 ORF1244 alanyl-tRNA synthetase 159, 160 cytoplasmic alaS PAC13 ORF0835 RNA processing enzyme/ATP-bdg. 161, 162 cytoplasmic ymfA PAC15 ORF1124 lipoamid acyltransferase component of 163, 164 cytoplasmic bfmBB branched-chain alpha-keto acid dehydrogenase complex PAC16 ORF0340 glyceraldehydes-3-phosphate cytoplasmic GAPDH dehydrogenase PAC17 not found 5′-methylthioadenosine nucleosidase/ cytoplasmic Contig83 S-adenosylhomo-cysteine nucleosidase PAC20 ORF2711 75% identity to ORF2715 167, 168 unknown similar to hypothetical proteins POV31 ORF0659 29 kDa surface protein 236, 238 surface POV32 ORF0659 29 kDa surface protein 236, 238 surface POV33 ORF0659 29 kDa surface protein 236, 238 surface POV34 ORF0659 29 kDa surface protein 236, 238 surface POV35 ORF0659 29 kDa surface protein 236, 238 surface POV36 ORF00661 LPXTG-motif cell wall anchor domain 235, 237 surface protein POV37 ORF0659 29 kDa surface protein 236, 238 surface POV38 ORF0659 29 kDa surface protein 236, 238 surface POV39 ORF0657 LPXTG-anchored surface protein 1/165,142 surface POV40 not identified Seq ID no: S. aureus ORF Putative antigenic surface areas (Protein) spot ID no./abbrev. Putative localization (Antigenic package) 112 PCK6 ORF2309 peripheral 61-75, 82-87, 97-104, 113-123, 128-133, mqo membrane 203-216, 224-229, 236-246, 251-258, 271-286, 288-294, 301-310, 316-329, 337-346, 348-371, 394-406, 418-435, 440-452 114 POV2 ORF766 aux1 trans-membrane 30-37, 44-55, 83-91, 101-118, 121-128, 136-149, 175-183, 185-193, 206-212, 222-229, 235-242 116 POV4 ORF078 EF-Tu cytoplasmic/ 28-38, 76-91, 102-109, 118-141, 146-153, secreted 155-161, 165-179, 186-202, 215-221, 234-249, 262-269, 276-282, 289-302, 306-314, 321-326, 338-345, 360-369, 385-391 176 PAC2 ORF1931 periplasmic 29-44, 74-83, 105-113, 119-125, 130-148, ModA 155-175, 182-190, 198-211, 238-245 174 PAC1 ORF1490 periplasmic 5-24, 38-44, 100-106, 118-130, 144-154, prsA 204-210, 218-223, 228-243, 257-264, 266-286, 292-299 168 PAC20 ORF2711 unknown 7-14, 21-30, 34-50, 52-63, 65-72, 77-84, 109-124, 129-152, 158-163, 175-190, 193-216, 219-234 GI no. or S. aureus protein Seq ID no: spot ID TIGR no. (ORF no./abbrev.) Putative function (by homology) (DNA, Prot) PCK2 TIGR1280 ORF0599 Glycinamide-ribosyl synthase 107, 108 PCK4 7672993 ORF2268 lsaA possibly adhesion/aggregation 12, 64 PCK5 TIGR6209 ORF0484 yitU conserved hypoth. protein (yitU) 109, 110 PCK6 TIGR6182 ORF2309 membrane-associated malate-quinone 111, 112 oxidase POV2 6434044 ORF0766 aux1 protein phosphatase contributing to methicilin 113, 114 resistance POV3.1 7672993 ORF2268 lsaA possibly adhesion/aggregation 12, 64 POV3.2 7672993 ORF2268 lsaA possibly adhesion/aggregation 12, 64 POV4 TIGR8079 ORF0078 EF- C-terminal part of 44 kDa protein similar 115, 116 Tu to elongation factor Tu POV5¹⁾ TIGR8091 ORF0782 3-ketoacyl-acyl carrier protein reductase 117, 118 (fabG) POV7 2500720 ORF0317 SecA protein transport across the membrane 39, 91 SecA POV10 TIGR8097 ORF1252 yrzC hypothetical BACSU 11.9 kd protein 119, 120 (upf0074 (rff2) family) POV12 2499415 ORF0621 pdhB dihydrolipoamide acetyltransferase (pdhB) 121, 122 POV13 7470965 ORF0094 SdrD fibrinogen-bdg. (LPXTG) protein homolog 123, 124 (SdrD) POV14 1350849 ORF0072 rpoB DNA-directed RNA polymerase β 125, 126 POV15 6920067 ORF0077 EF-G 85 kD vitronectin binding protein 127, 128 POV17 TIGR8079 ORF0078 C-terminal part of 44 kDa protein similar 115, 116 to elongation factor Tu POV18 3025223 not found general stress protein YLY1 129, 130 POV30¹⁾ 350771 ORF0069 RL7 ribosomal protein L7 131, 132 POV21 ORF0103 probable hexulose-6-phosphate synthase 133, 134 (yckG) POV23 ORF0182 lipoprotein (S. epidermis) 135, 136 ¹⁾identified from a total lysate from S. aureus 8325-4 spa-grown under standard conditions. Seroreactivity with 1/1 patient and 2/4 normal sera but not with infant serum (C5).

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1. A method for identifying hyperimmune serum reactive antigens of a pathogen comprising: providing an antibody preparation from a plasma pool of a given type of animal or from a human plasma pool or individual sera with antibodies against a specific pathogen; providing at least one genomic frame-selected bacterial FhuA surface expression library of said specific pathogen; screening said at least one genomic frame-selected bacterial FhuA surface expression library with said antibody preparation; identifying antigens which bind in said screening to antibodies in said antibody preparation; screening the identified antigens with individual antibody preparations from individual sera from individuals with antibodies against said specific pathogen, wherein the individuals have an antibody titer against said specific pathogen of higher than 90^(th) percentile and an IgG titer above 10000 U; and identifying hyperimmune serum-reactive antigens that bind to a portion of said individual antibody preparations from said individual sera.
 2. The method of claim 1, further comprising: providing at least a second and a third different expression library of said specific pathogen, screening said at least a second and a third different expression library with said antibody preparation, identifying antigens which bind in at least one of said screenings of said genomic frame-selected bacterial FhuA surface expression library, said second different expression library, and said third different expression library to antibodies in said antibody preparation, screening the identified antigens with individual antibody preparations from individual sera from individuals with antibodies against said specific pathogen, wherein the individuals have an antibody titer against said specific pathogen of higher than 90^(th) percentile and an IgG titer above 10000 U, identifying hyperimmune serum-reactive antigens that bind to a portion of said individual antibody preparations from said individual sera, repeating said screening and identification steps at least once, comparing the hyperimmune serum-reactive antigens identified in the repeated screening and identification steps with the hyperimmune serum-reactive antigens identified in the initial screening and identification steps, further repeating said screening and identification steps, if at least 5% of the hyperimmune serum-reactive antigens have been identified in the repeated screening and identification steps only, until less than 5% of the hyperimmune serum-reactive antigens are identified in a further repeating step only to obtain a complete set of hyperimmune serum-reactive antigens of a specific pathogen.
 3. The method of claim 2, wherein said at least second and third different expression libraries are at least a ribosomal display library, a bacterial surface library and a proteome.
 4. The method of claim 1, wherein said plasma pool is a human plasma pool taken from at least one individual having experienced or are experiencing an infection with said pathogen.
 5. The method of claim 1, wherein said genomic frame-selected bacterial FhuA surface expression library of said pathogen has a redundancy of at least 5×.
 6. The method of claim 1, further defined as comprising screening at least a ribosomal display library, a bacterial surface display library and a proteome with said antibody preparation and identifying antigens which bind in at least two of said screenings to antibodies in said antibody preparation.
 7. The method of claim 1, wherein said pathogen is a bacterial, viral, fungal or protozoan pathogen.
 8. The method of claim 1, wherein said pathogen is a Staphylococcus.
 9. The method of claim 1, further comprising isolating identified hyperimmune serum-reactive antigens.
 10. The method of claim 1, further comprising producing isolated hyperimmune serum-reactive antigens by chemical or recombinant methods.
 11. The method of claim 10, further comprising formulating the produced hyperimmune serum-reactive antigens in a pharmaceutical preparation.
 12. The method of claim 1, wherein said antibody preparation is derived from at least one patient with acute infection with said pathogen.
 13. The method of claim 1, wherein at least 10 individual antibody preparations are used in identifying said hyperimmune serum-reactive antigens.
 14. The method of claim 1, wherein said hyperimmune serum-reactive antigens bind to at least 10 of said individual antibody preparations used in said screening.
 15. The method of claim 1, wherein said hyperimmune serum-reactive antigens bind to at least 20% of said individual antibody preparations used in said screening.
 16. The method of claim 1, wherein individual sera are selected which have an IgA titer against a lysate, cell wall components, or recombinant proteins of said pathogen above 4000 U. 